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Full text of "Change in dehydrodolichyl diphosphate synthase during spermatogenesis in the rat"

CHANGE IN DEHYDRODOLI CHYL DIPHOSPHATE SYNTHASE 
DURING SPERMATOGENESIS IN THE RAT 



BY 

ZHONG CHEN 



DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE 
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE 
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 



1988 



This dissertation is dedicated to my 
parents, Quo-Wei Chen and Zhi-Huei Chang, 
my loving wife, He-Ping Han, and my son, 
Henry Hai Pei. 



ii 



ACKNOWLEDGEMENTS 



Sincere appreciation is expressed to my friend, mentor, 
Dr. Charles M. Allen, Jr., for his enduring patience, 
guidance, and support. I am deeply grateful for his help in 
proofreading my English. His belief in the fundamentals of 
biochemistry and molecular biology and literacy influenced 
the designing of each project undertaken, and I learned the 
meaning of scientific integrity, creativity, and serendipity. 

I am indebted to the members of my supervisory 
committee, Dr. Michael S. Kilberg, Dr. Rusty J. Mans, Dr. 
Thomas W. O'Brien, and Dr. Lynn J. Romrell for their 
encouragement, assistance and productive criticism of my 
research projects. I am especially grateful to Dr. Lynn J. 
Romrell who allowed me the privilege of working in his 
laboratory and who provided support and advice through the 
duration of the endeavor. 

In addition, I am indebted to the faculty of the 
Department of Biochemistry and Molecular Biology for both 
educational and financial support. Finally, I would like to 
thank William Blakeney, Mary Handlogton, Michael Campa and 
William Wong for their friendship and assistance during the 
course of my studies. 



iii 



TABLE OF CONTENTS 



Page 



ACKNOWLEDGEMENTS tii 

LIST OF TABLES vi 

LIST OF FIGURES vii 

ABBREVIATIONS ix 

ABSTRACT xi 

CHAPTERS 

I INTRODUCTION 1 

The Spermatogenesis 1 

The Sertoli Cells 7 

The Role of DOL P in Glycoprotein B i o synthe s i s . . . . 1 2 
Studies Related to DOL Biosynthesis and 

Spermatogenesis 26 

S i gni f i cane e 3 5 

Obj ectives 37 

II DEVELOP AND OPTIMIZE AN ASSAY FOR DEHYDRO 

DOLICHYL DIPHOSPHATE SYNTHASE FROM RATS TESTES.... 38 

Introduction 38 

Materials and Methods 41 

Results 46 

Discussion 83 

III DEHYDRODOLI CHYL DIPHOSPHATE SYNTHASE ACTIVITY 
MEASURED IN ENRICHED SPERMATOGENIC CELL 
POPULATIONS 93 

Introduction 93 

Materials and Methods 98 

Results 103 

Discuss ion 122 

IV CONCLUSIONS AND DIRECTIONS 131 



iv 



APPENDICES 



A SUMMARY OF EXPERIMENTAL DATA PRESENTED IN 

FIGURE 2-12. DEHYDRO DOL PP SYNTHASE ACTIVITY 



IN SONICATES OF TUBULES FROM RATS OF 

DIFFERENT AGES 134 

B TYPICAL NUMBER OF RATS, TOTAL TUBULE WEIGHT, 
AND NUMBER OF ASSAY USED FOR EACH EXPERIMENT 
AS A FUNCTION OF RAT AGE 135 

C SUMMARY OF EXPERIMENTAL DATA PRESENTED IN 
FIGURE 3-5. AGE DEPENDENT VARIATION IN 
SYNTHASE ACTIVITY IN SERTOLI CELLS, 
SPERMATOGENIC CELLS AND PROTEASE TREATED 
SEMINIFEROUS TUBULES 136 

BIBLIOGRAPHY 137 

BIOGRAPHICAL SKETCH 146 



v 



LIST OF TABLES 



Table Page 

2-1 Incorporation of A 3 -[1- 14 C] Isopentenyl Diphosphate 
and [a, /3- 32 P] - Isopentenyl Diphosphate into 
Dehydro DOL PP and Dehydro DOL P 52 

2- 2 Formation of Enzymatic Product at Different Triton 

X-100 Concentrations in Pulse-Chase Experiment 58 

3- 1 Dehydro Dolichyl Diphosphate Synthase Activity in 

Enriched Spermatogenic Cells 117 

3-2 Estimated Specific Activities of Dehydro DOL PP 

Synthase in Pure Spermatogenic Cells 120 

3-3 Estimated Specific Activities of Dehydro DOL PP 
Synthase in "Pure" Sertoli Cells From Rats of 
Different Ages 121 



vi 



LIST OF FIGURES 

Fi gure Page 

1-1 The Cellular Composition of the 14 Stages of the 

Cycle of the Seminiferous Epithelium in Rat 4 

1-2 Schematic Drawing of Human Seminiferous Ep i the 1 ium . . . . 9 

1-3 DOL Cycle for Glycoprotein Formation in 

Eucaryotic Cells 16 

1-4 A Putative Pathway Showing the Relationship between 

Dehydro DOL PP and Glycoprotein Biosynthesis 22 

1-5 Pathway of Isoprenoid Biosynthesis 25 

1- 6 The Structure of Dehydro DOL PP and DOL PP 28 

2- 1 Separation of Enzymatic Products by TLC 49 

2-2 Triton X-100 Dependency on the Formation of 

Dehydro DOL PP and Dehydro DOL P 53 

2-3 Dependence of Product Formation on Triton X-100 

Concentration and Incubation Time 56 

2-4 Time Course of Dehydro DOL PP and Dehydro DOL P 

Formation 61 

2-5 Product of Base Hydrolysis of Dehydro DOL PP 63 

2-6 Effect of Protein Concentration on Dehydro DOL PP 

and Dehydro DOL P Formation 67 

2-7 Isopentenyl Diphosphate Concentration Dependency on 

the Formation of Dehydro DOL PP and Dehydro DOL P.... 69 

2-8 A Double Reciprocal Plot of the Sum of Dehydro 

DOL PP and Dehydro DOL P Formation vs. Isopentenyl 
Diphosphate Concentrations 71 

2-9 Farnesyl Diphosphate Concentration Dependency on 

the Formation of Dehydro DOL PP and Dehydro DOL P.... 73 



vii 



2-10 A Double Reciprocal Plot of the Sum of Dehydro 
DOL PP and Dehydro DOL P Formation vs. Farnesyl 
Diphosphate Concentration 75 

2-11 Time Course of Incorporation of [ -^C ] - Isopentenyl 

Diphosphate into Dehydro DOL PP and Dehydro DOL P.... 77 

2-12 Dehydro DOL PP Synthase in Testicular Homogenate 

of Different Aged Rats 79 

2-13 Dehydro DOL PP Synthase Activity in Sonicates of 

Tubules from Rats of Different Ages 81 

2- 14 Comparison of Changes in DOL P Concentration, 

and Dehydro DOL PP Synthase Activity as a 

Function of Rat Age 88 

3- 1 Purity of Enriched Cell Fractions 105 

3-2 Dehydro Dolichyl Diphosphate Synthase Activity in 

Sonicates of Tubules from Rats of Different Ages.... 109 

3-3 Time Course of Incorporation of [ 14 C] -Isopentenyl 

Diphosphate into Dehydro DOL PP and Dehydro DOL p...lll 

3-4 Dehydro Dolichyl Diphosphate Synthase Activity in 

Enriched Spermatogenic Cell Population 115 

3-5 Age Dependent Variation in Synthase Activity in 
Sertoli Cells, Spermatogenic Cells and Protease 
Treated Seminiferous Tubules 118 

3-6 The Relationship between the Dehydro DOL PP 
Synthase Activity and Spermatogenesis during 
Testicular Development 128 



viii 



KEY TO ABBREVIATIONS 



BSA bovine serum albumin 

Ci curie 

cm centimeter 

cpm counts per minute 

°C degree centigrade 

DIBK diisobutyl ketone 

DNA deoxyribonucleic acid 

DNase deoxyr ibonuclease 

DOL dolichol 

DOL P dolichyl phosphate 

DOL PP dolichyl diphosphate 

dpm disintegrations per minute 

EKRB enriched Krebs-Ringer bicarbonate medium 

IPP isopentenyl diphosphate 

FPP t , t-farnesyl diphosphate 

Glc glucose 

GlcNAc acetylglycosamine 

GPP geranyl diphosphate 

g gram 

HAc acetic acid 

hr hour 

M molar 



ix 



Man mannose 

MEM Eagle's minimal essential medium 

H micron 

fid microcurie 

fig microgram 

fil microliter 

^M micromolar 

/imole micromole 

mM milliraolar 

mm millimeter 

mmole millimole 

PBS phosphate buffered saline 

pmole picoraole 

PP diphosphate 

RNA ribonucleic acid 

rpm revolutions per minute 

TLC thin layer chromatography 

Tris Tris-(hydroxymethyl) am inome thane 

v/v on a volume - to -volume basis 

v/w on a volume-to-weight basis 

w/v on a we i gh t - t o - vo lume basis 

w/w on a we igh t - to - we ight basis 



x 



Abstract of Dissertation Presented to the Graduate School of 
the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 



CHANGE IN DEHYDRODOLI CHYL DIPHOSPHATE SYNTHASE 
DURING SPERMATOGENESIS IN THE RAT 



by 



ZHONG CHEN 



April, 1988 



Chairman: Charles M. Allen, Jr. Ph.D. 

Major Department: Biochemistry and Molecular Biology 



Dolichyl phosphate (DOL P) , a carbohydrate carrier in 
glycoprotein biosynthesis, may be formed from the metabolism 
of dehydro dolichyl diphosphate (dehydro DOL PP). A method 
has been developed to measure 2,3-dehydro DOL PP synthase 
with [ * ^ C ] - i s o p en t eny 1 diphosphate and t,t-farnesyl 
diphosphate. Enzymatic activity was measured in homogenates 
prepared by sonication of seminiferous tubules or isolated 
cell fractions. There was a 2 fold increase in tubular 
activity between day 7 and day 23 and a similar decrease in 
activity between day 23 and day 60. The increase in activity 
paralleled an increase in DOL P concentration, suggesting a 
regulatory role for the synthase in DOL P synthesis. 



xi 



The activity in homogenates of protease treated 
seminiferous tubules, and cell fractions enriched in 
spermatogenic cells or Sertoli cells also changed as a 
function of age in the rats. The highest enzymatic activity 
occurred in each case at age 23 days. Cell fractions 
enriched in pachytene spermatocytes, spermatids or Sertoli 
cells were shown to have higher synthase activity than a 
whole testicular homogenate or a mixture of cells prepared by 
col lagenase - tryps in treatment of tubules. Enzymatic activity 
in pachytene spermatocytes expressed as pmoles/mg protein was 
about 5.3 fold higher than spermatogonia, 1.7 fold higher 
than spermatids and about 8.3 fold higher than spermatozoa. 
The enzymatic activity of pachytene spermatocytes expressed 
as pmoles/10^ cells was 4.5 fold higher than spermatids and 
about 126 fold higher than spermatozoa. These studies are 
the first to show that Sertoli cells have the potential to 
synthesize DOL. 

The increase in synthase activity in spermatogenic 
cells and Sertoli cells during early stages of 
spermatogenesis indicates that both cell populations are 
contributing to the increase in activity in seminiferous 
tubules. Furthermore, the increase can be explained on the 
basis of changes in the specific activities of the Sertoli 
cells and the different populations of spermatogenic cells. 
This increase may be important in regulating the availability 
of dolichyl phosphate for glycoprotein biosynthesis during 
early stages of spermatogenesis. 

xii 



CHAPTER I 
INTRODUCTION 



Spermatogenesis 



Spermato genie Cycles and Waves 

Spermatogenesis is a developmental process in which the 
spermatogenic cell undergoes a series of biochemical and 
morphological changes through three well described phases: 
spermatogonial renewal and proliferation, meiosis, and 
spermiogenesis . Spermatogenesis in the rat starts during 
fetal development with appearance of gonocytes by postnatal 
day 4 and continues throughout adult life. The first 

spermatozoa appear within the lumens of the seminiferous 
tubules at about 45 days of ages, and all stages of 
spermatogenic cycle are represented (Clermont & Perey, 1957; 
Knorr et al. , 1970). The initial phase of spermatogenesis, 
the spermatogonial phase, occurs in the basal compartment of 
the seminiferous epithelium and consists of a mitotic 
proliferation of spermatogonia from stem cells. The 
spermatogonia divide and differentiate sequentially into type 
A spermatogonia, intermediate spermatogonia, and type B 
spermatogonia. The type B spermatogonia divide to form 



2 

preleptotene primary spermatocytes, which undergo a final 
replication of nuclear DNA before entering meiotic prophase. 
The preleptotene spermatocytes migrate from the basal 
compartment to the adluminal compartment where 
spermatogenesis is completed. The second phase of 

spermatogenesis, meiosis, occurs while the spermatocytes 
remain on the adluminal side of the intercellular Sertoli 
junctions. Meiotic prophase, which is subdivided into the 
stages of the leptotene, zygotene, pachytene, diplotene , and 
diakinesis, terminates in the first meiotic, or reductional, 
division with the formation of secondary spermatocytes. The 
latter cells quickly enter the second meiotic, or equational 
division to form the haploid spermatids. Spermiogenes is , the 
final phase of spermatogenesis, consists of a complex 
morphological transformation of the haploid spermatogenic 
cell, that culminates with the release of late spermatids 
into the lumen of the seminiferous tubule. 

Spermatogenesis has many unique features. The most 
remarkable ones include the spermatogenic cycles and waves in 
the seminiferous epithelium. The various generations of 
spermatogenic cells are not randomly distributed in the 
seminiferous epithelium, but are organized into well-defined 
cellular associations. Certain cells are always found in 
association with certain other cells. Each of these cells 
develops in synchrony with the others, so that if we could 
watch one section of the tubule wall with a time-lapse 



3 

camera, a series of different cell associations would be 
seen, until the cycle was completed. The time interval 
between the appearance of the same cell association at a 
given point of the tubule is called the cycle of the 
seminiferous epithelium. The number of stages in the cycle 
is constant for a given species; man has 6, mouse and monkey 
have 12, the rat, 14 (Figure 1-1). 

The cycle involves changes with time in the appearance 
of one segment of a tubule, whereas the wave refers to the 
distribution of different cellular associations along the 
length of the tubule. The segments of the tubules which 
specific cellular associations occur in a sequential series 
along the length of the tubule. For example a segment 
containing cells at stage V of the cycle is bordered on one 
side by cells at stage IV and on the opposite side is at 
stage VI. The average length of each tubular segment 

correlates roughly with the relative duration of the 
corresponding cellular association or stage of the cycle. 
The sequence of segments or waves, representing the stages in 
the cycle of the seminiferous epithelium, repeats itself 
along the length of an individual tubule. In the rat, for 
example, there is an average of 12 waves per tubule. 



4 



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STAGES OF THE CYCLE 



Figure 1-1. The Cellular Composition of the 14 Stages 
of the Cycle of the Seminiferous Epithelium in Rat. 

Each numbered column (roman numeral) shows the 
spermatogenic cell types present in cellular associations 
found in cross sections of seminiferous tubules. The 
cellular associations or stages of the cycle succeed one 
another in time in any given area of the seminiferous 
epithelium in the rat. Following cellular association XIV, 
cellular association I reappears, so that the sequence start 
over again. Steps in the development of the spermatids, 
numbered 1 to 19 and defined by changes in the structure of 
the nucleus and acrosome, are indicated with arable numerals. 
Letters are used to identify spermatogonia and spermatocytes. 
A]_, A 2 , A3, and A4 , represent four generations of type A 
spermatogonia; In, intermediate spermatogonia; B, type 
spermatogonia; PI, preleptotene spermatocytes; L, leptotene 
spermatocytes; Z, zygotene spermatocytes; P, pachytene 
spermatocytes; Di , Diplotene and Diakinesis; II, secondary 
spermatocytes. The subscript m indicates the occurrence of 
mitotic division of the spermatogonia (From Dym, M. & 
Clermont, Y., 1970, Am. J. Anat., 128, 265-282,). 



5 

Exocrine and Endocrine Functions of Testis 

The testis has both exocrine and endocrine functions. 
The exocrine function of the testis resides in the cells of 
the seminiferous epithelium which produce testicular fluids 
and spermatozoa. The endocrine function of the testis 
resides primarily in the Leydig cell population which 
synthesizes and secretes the principal circulating androgen, 
testosterone (Nearly 95% of the testosterone is produced by 
the testis; the rest is produced by the adrenal glands). The 
steroidogenic and spermatogenic activities of the testis are 
regulated by hormonal interactions among the hypothalamus, 
adenohypophysis , and the cells of the gonad — the Sertoli 
cells, spermatogenic, and Leydig cells. 

The Leydig cells, which are located in the space 
surrounding the seminiferous tubules, start to secrete 
testosterone during the fetal period, promoting the 
development of the male reproductive tract. 

Biosynthesis of Glycoproteins During Spermatogenesis in 

Testis 

Mammalian spermatogenesis involves extensive 
morphological and biochemical transformations to produce 
mature gametes that are structurally and functionally unique. 
During this process, gene expression is temporally regulated 
both at transcriptional and translational levels (Bellve & 
O'Brien, 1983; Hecht, 1986). A variety of proteins, many of 
which are unique to spermatogenic cells, are differentially 



expressed during meiosis and spermiogenes is . These include 
basic chromosomal proteins that undergo successive 
transitions during s p e r m a t o g e n i c cell differentiation 
(Meistrich et al., 1981), several spermatogenic cell - spec if ic 
isozymes (Goldberg, 1977) and s t a g e - s p e c i f ic surface 
glycoproteins identified with biochemical (Millette & 
Moulding, 1981a; Millette & Moulding, 1981b) and 
immunological probes (O'Rand & Romrell, 1980; Gaunt, 1982; 
Fenderson et al . , 1984; O'Brien & Millette, 1984; O'Brien & 
Millette, 1986). It has been suggested that many proteins 
play roles in various processes including sperm-egg 
interaction, S e r t o 1 i - s p e r ma t o ge n i c cell association, 
capacitation and the acrosomal reaction. For example, rabbit 
sperm autoantigen (RSA-1), a s ialoglycoprote in located in the 
postacrosomal region, plays a role in the spermatozoon's 
binding to and penetration of the zona . This protein first 
appears on the surface of pachytene spermatocytes and 
increases in amount throughout spermatogenesis (O'Rand & 
Romrell, 1981; O'Rand et al . , 1984). Such sperm specific 
surface components are probably involved in antibody mediated 
agglutination and immobilization of spermatozoa. 

Early in spermatogenesis, glycoproteins are synthesized 
and deposited in the acrosome. One of the acrosomal 

glycoproteins is proacrosin, which yields acrosin after being 
activated. Proacrosin is first produced during the spermatid 
stage of differentiation, and is retained throughout the 



remainder of spermatogenesis (Florke et al . , 1983). Acrosin 
is thought to be an essential protease required for the 
proteolysis of the zona pellucida of the ovum during 
fertilization (Hartree, 1977). In purified rabbit 

proacrosin, glucosamine, mannose, galactose and sialic acid 
were found in the ratio of 3,3,1,1 per mole of proacrosin 
which is consistent with the ratio expected of these sugars 
in N-linked glycoproteins (Mukerji & Meizel, 1979). 
Therefore, proacrosin would be expected to be synthesized via 
a pathway which involved dolichyl phosphate (DOL P) 
metabolism . 

A general study of the glycosy lat ion of protein would 
be of value in understanding factors which might regulate the 
biosynthesis of these cell and stage specific proteins. An 
understanding of the origin, metabolic pathway and mechanism 
of biosynthesis of these glycoproteins during spermatogenesis 
may provide insight into the biochemical control of the cell 
function and differentiation. These findings may be of 
significance in the design of male contraceptive agents and 
in understanding molecular basis of male sterility. 

Sertoli Cell 



Histological Structures and Functions 

The Sertoli cells are the nongerminal elements 
seminiferous tubules of the testes. They were 



in the 



first 



8 

described in 1865 by Sertoli to have a nursing function, to 
provide mechanical support for the developing spermatogenic 
cells as well to be phagocytic. It is believed that 

circulating hormones, which act on spermatogenesis, have 
their effects mediated via the Sertoli cell (Hansson et al . , 
1976) . 

Sertoli cells are basically columnar cells, which 
surround the adjacent spermatogenic cells and fill the spaces 
between them (Figure 1-2). Sertoli cells form the major 
structural component of the seminiferous tubules and serve a 
number of functions, including (a) mediating movement of 
spermatogenic cells from the basal lamina to the lumen and 
the release of the late spermatids into the tubular lumen, 
(b) compartmentalizing the epithelium into basal and 
adluminal compartments and forming part of the blood-testis 
barrier, (c) phagocy tiz ing degenerating spermatogenic cells 
and residual bodies, (d) secreting androgen binding protein 
and inhibin as well as other glycoproteins and (e) mediating 
the movement of steroids, metabolites, and nutrients utilized 
by spermatogenic cells across the seminiferous epithelium. 

During development, the Sertoli cell undergoes 
fundamental changes. The most remarkable feature of these 
maturational changes is the cessation of cell multiplication 
that occurs before puberty. It occurs in the rat at about 15 
days of age (Steinberger & Steinberger, 1971). After that 
age, the Sertoli cells may change their metabolic activities 



9 




Figure 1-2. Schematic Drawing of Human Seminiferous 
Epithelium . 

The seminiferous epithelium recline upon a basal lamina 
( BL) , and a layer of peritubular cells (PT) surrounds the 
seminiferous tubule. Pale type A spermatogonium (Ap), dark 
type A spermatogonium (Ad) , and type B spermatogonium are 
located in the basal compartment of the seminiferous 
epithelium below the junctional complex (JC) between adjacent 
Sertoli cells (SC); pachytene primary spermatocytes (P), 
early spermatids (ES), and late spermatids (LS) are seen in 
the adluminal compartment above the junctional complex (Ross, 
M. H. and Reith, E. J., 1985, Histology, 3rd printing, pp 
608; Clermont, Y. 1963, Am. J. Anat. , 112, 35). 



10 

under the influence of various factors, but they do not 
divide any more. 

Adjacent Sertoli cells are joined by the Sertoli cell 
junctional complex, which is a unique structure not found in 
other epithelium tissues. This functional barrier develops 
in the rat at about 16 to 19 days of age (Vitale et al . , 
1973). The S er tol i - S er to 1 i junctional complex divides the 
seminiferous epithelium into two compartments: the basal 
compartment and the adluminal compartment. Actually, the 
Sertoli - Sertoli junctional complexes are the site of the 
blood-testis barrier, which serves an essential role in 
isolating the spermatogenic cells from the immune system; 
i.e. the production of unique molecules on spermatogenic 
cells is recognized as foreign if these molecules come in 
contact with the immune system. 

Follicle stimulating hormone (FSH) and testosterone 
regulate the process of sperm production within the 
seminiferous epithelium. The Sertoli cells have been shown 
to be the primary target for FSH and androgens. Therefore, 
the Sertoli cells are considered to be the regulators of 
spermatogenesis. The probable importance of Sertoli cells in 
spermatogenesis has been emphasized by a number of 
investigators (Bridges et al . , 1986; Fritz et al . , 1976; 
Griswold et al . , 1986); however, their precise role in this 
process is still not fully understood. 



11 

Physiological and morphological studies have indicated 
that the Sertoli cells undergo cyclic changes in their 
metabolic activity which are related to specific stages in 
the cycle of the seminiferous epithelium. Cytochemical 
studies showed that several enzyme activities in Sertoli 
cells vary depending on the stage of the cycle of the 
seminiferous epithelium. For instance, the peak activity of 
acid phosphatase appeared at stages VII and VIII, but little 
activity was found in stages IX through II (Niemi & Kormano, 
1965) . A similar stage specific distribution has been 
observed with the thiamine pyrophosphatase in Sertoli cells 
(Hilscher et al . , 1979) . 

Sertoli Cells Secrete Many Glycoproteins 

One of the mechanisms by which Sertoli cells 
biochemically influence the spermatogenic cells is through 
the synthesis and secretion of glycoproteins. At least 15% 
of all the proteins synthesized by Sertoli cell are 
glycoproteins (Bridges et al., 1986). Some of the 

glycoproteins secreted by the Sertoli cells are specific to 
the testis, and others are similar, if not identical, to 
serum proteins. Sertoli cells secrete glycoproteins into the 
lumen of the tubules, perhaps into the blood stream or lymph, 
and maybe into the space between Sertoli cells and 
spermatogenic cells for subsequent uptake by spermatogenic 
cells. Several glycoproteins secreted by the Sertoli cells 



12 

have been studied, such as androgen binding protein (ABP) 
(Fritz et al . , 1976), plasminogen activator (Lacroix et al . , 
1977), testicular transferrin (Skinner & Griswold, 1980), and 
sulfated glycoprotein 1 (SPG-1) and 2 (SPG-2) (Griswold et 
al., 1986). 

The stage specific nature of the elaboration of one 
glycoprotein, androgen binding protein, has been particularly 
well described. Sertoli cells have maximum FSH binding 
during stages XIII through I followed by maximum cAMP 
production in stages II through VI which in turn is followed 
by maximum production of androgen binding protein in stages 
VII through VIII (Parvinen et al . , 1980; Ritzen et al . , 
1982). Since Sertoli cells are actively involved in 

glycoprotein biosynthesis, we speculate that the regulation 
of the N-linked glycoprotein biosynthesis may be significant 
in the function of these cells. 

Role of POL P in Glycoprotein Biosynthesis 

The Structure and Distribution of Dolichol and Its 
Derivatives 

Dolichol (DOL) is a general term for a group of 
polyisoprenoid alcohols. They contain the dimethylallyl 
terminal unit (w- terminal ) , two trans - isoprene residues, a 
number of cis - isoprene residues and a terminal hydroxylated 
a-saturated isoprene unit linked in a head-to-tail 
orientation. Pennock, Hemming and Morton first isolated and 



13 

characterized this compound from pig liver (Pennock et al. , 
1960) . Mammalian polyprenols contain a larger number of 
isoprene residues C85-C115 (17-23 x C 5 ) than those present in 
plants and bacteria C 50 -C 60 (10-12 x C 5 ). Polyprenols found 
in bacteria have an a- unsaturated isoprene unit. However, it 
is known that liver contains small amounts of a-saturated 
shorter polyprenols (Mankowski et al., 1976), and in some 
tissues, such as bovine pituitary gland ( Rodominska - Pyr ek et 
al., 1979) and hen oviduct (Hayes & Lucas, 1980), a- 
unsaturated compounds have been reported. DOL is present in 
most eukaryotic cells and is found in particularly high 
concentration in some human tissues, such as the adrenal 
gland, pancreas, pituitary gland, testis and thyroid gland 
(Rupar & Carroll, 1978; Tollbom & Dallner, 1986). James and 
Kandutsch showed that in mouse the synthesis of DOL is much 
more active in testes than in liver (James & Kandutsch, 
1980c) . It seems that DOL content is relatively high in the 
rapidly growing and differentiating tissues. For example, 
the DOL content of the hyperplastic liver nodules in liver is 
four times higher in the homogenate and six times higher in 
the microsomes than that found in normal rat liver. In 
developed hepatocarc inoma , the amount of DOL was found to be 
doubled. In contrast to free DOL, DOL P was found to be 
greatly decreased in nodules (Eggens et al . , 1984). 

Other studies show an increase in DOL levels with age 
although the increases vary widely with tissues (brain, 



14 

liver, kidney, testis, lung and heart) (Pullarkat et al . , 
1984) . 

DOL may have some effects on membrane structure and 
fluidity (Valtersson et al . , 1985). Otherwise, most DOL 
apparently has no direct relationship to glycoprotein 
synthesis, since the bulk of the DOL is present in membranes 
other than the endoplasmic reticulum (Wong et al . , 1982; 
Adair & Keller, 1982; Ekstrom et al . , 1982). Some DOL may be 
phosphorylated by a CTP - dependent kinase (Allen et al . , 1978; 
Burton et al . , 1979). Dolichol is found in several combined 
forms in the cell, free dolichol and esterified form with 
fatty acids or mono or diphosphate. The phosphorylated forms 
are important as we will see soon because they are also found 
with different degrees of glycosylat ion which is important 
for glycoprotein biosynthesis. 

Dolichyl fatty acyl esters may have several metabolic 
roles. First, the fatty acyl esters may be a suitable and 
stable form for the storage of dolichol in lipid droplets. 
Alternatively, the fatty acyl moiety might be necessary for 
the transport of dolichol from its site of synthesis to 
different subcellular locations. 

The percentage of dolichol found in the esterified form 
varies widely in different tissue. For example, it is 
reported to be 0% and 25% esterified in pig kidney and 
spleen, respectively. In another report, it is shown that 
the ester form represents about 63% of total DOL in pig 



15 

liver, and 65-90% in mouse testes and preputial glands 
(Malvar et al . , 1985). The mechanism which determines these 
distributions is not known. 

POL P in Glycoprotein Synthesis 

In the early 1970s, Behrens and Leloir demonstrated the 
involvement of DOL P in the N-linked glycoprotein 
biosynthesis (Behrens & Leloir, 1970; Behrens et al . , 1971). 
Although, only a small amount of the total DOL in cells is 
phosphorylated (Eggens et al . , 1983), the phosphorylated form 
(DOL P) is an essential carbohydrate carrier in the 
biosynthesis of asparagine - 1 inked glycoproteins (Struck & 
Lennarz, 1980; Hubbard & Ivatt, 1981). 

Most secretory proteins and membrane proteins, numerous 
receptors and proteins related to cell recognition are 
glycoproteins. A typical N-linked glycoprotein contains one 
or a few oligosaccharide units linked to asparagine side 
chains by N-glycosidic bonds. 

The biosynthesis of N-linked glycoproteins proceeds 
through a cyclic process, which requires the involvement of 
DOL P, in the synthesis of the core oligosaccharide unit 
(Parodi & Leloir, 1979; Hubbard & Ivatt, 1981). 

Figure 1-3 represents a highly abbreviated 
representation of this cyclic process and associated 
reactions (Rip et al . , 1985; Hemming, 1985, for review). 



16 



UDPGlcNAc 




5 GDPMan 



GlcNAcP-P-Dol 4 GDPMan Man 5 GlcNAc 2 P-P-Dol 




Figure 1-3. Dolichol cycle for glycoprotein formation 
in eukaryotic cells. 



17 



The first step in the assembly of 1 ip id - 1 inked 
oligosaccharide involves the addition of GlcNAc-P from UDP- 
GlcNAc to DOL P to generate DOL PP-GlcNAc. Then, this 
molecule reacts with an additional UDP-GlcNAc to form DOL PP- 
(GlcNAc)2- Five mannose residues are added next from GDP - 
mannose to form DOL PP - ( G lcNAc ) 2 - Man 5 . It had been shown 
that DOL P-Man also is a mannose donor to the core 
oligosaccharide chain. In the oligosaccharide lipid carrying 
nine mannose residues, the last four of these are transferred 
through DOL P (Rearick et al . , 1981). The next steps are 
transfers of three glucose units from DOL P-Glc to the core 
oligosaccharide chain with the formation of the lipid-linked 
oligosaccharide DOL PP - ( GlcNAc ) 2 - Man 9 - Glc 3 . In the final 
step of this pathway, the core oligosaccharide is transferred 
en bloc to newly synthesized polypeptide with the concomitant 
release of DOL PP. This core protein linked oligosaccharide 
is then processed by a now well described pathway involving 
specific glycosidases (Kornfeld & Kornfeld, 1980) and 
additional sugar residues added by DOL P independent glycosyl 
transferases. Schachter and his coworkers from studies of 
glycoprotein and glycolipid metabolism during spermatogenesis 
in rat and mouse testis indicated that spermatocytes and 
early spermatids had highly active glycosylating systems 
(Kornblatt et al . , 1974; Letts et al . , 1974a; Letts et al . , 
1974b) . 



18 

DOL P plays a major role in the biosynthesis of N - 
linked glycoprotein, since DOL P not only is an 
oligosaccharide unit carrier but also is an activator which 
reacts with certain nucleotide sugars and facilitates the 
sugar transfer to the core oligosaccharide chains. The 
involvement of DOL P in the production of the DOL PP- 
oligosaccharide and the subsequent transfer of the 
oligosaccharide to asparagine residues within the newly 
formed peptide is thought to occur on the luminal surface of 
the endoplasmic reticulum membrane (Pfeffer & Rothman, 1987, 
for review) . 

Studies with chick embryo fibroblast (Hubbard & 
Robbins, 1980) and canine kidney cells (Schmitt & Elbein, 
1979) suggest that oligosaccharide transfer is the rate 
limiting step in glycoprotein synthesis. Subsequent 
regeneration of DOL P permits the reinitiation of lipid 
oligosaccharide biosynthesis. Therefore, enzymes of the DOL 
P pathway associated with formation or utilization of DOL P 
could be very important in the control of glycoprotein 
biosynthesis in spermatogenesis. 

The availability of DOL P was found to be a rate- 
limiting factor in some glycosylation processes (Potter et 
al., 1981a; Eggens et al . , 1984). Furthermore, the shortage 
of the lipid intermediates influences some vital biological 
processes such as embryonic development (Carson et al . , 
1981). Carson and Lennarz showed that when DOL P synthesis 



19 

was inhibited by compactin, a potent inhibitor of 
hydroxymethyl glutaryl CoA reductase and consequently 
polyisoprenoid biosynthesis, protein glycosylation was 
impaired and the oligosaccharide chains synthesized were more 
negatively charged (Carson & Lennarz , 1981). In another 
report, inhibition of DOL P biosynthesis induced abnormal 
gastrulation in sea urchin embryos (Carson & Lennarz, 1979). 
All of the findings suggested that DOL P plays an important 
role in the N-linked glycoprotein biosynthesis. Needless to 
say, a good understanding in the regulation of DOL metabolism 
is a necessary step to explore the regulation of the N-linked 
glycoprotein biosynthesis. 

Metabolism and Functions of DOL P 

There are at least three pathways to generate DOL P. 
First, the dephosphorylation of DOL PP could provide the main 
supply of DOL P for the biosynthesis of intermediates in the 
protein glycosylation reactions (Dallner & Hemming, 1981). 
On the other hand, investigations in recent years have 
established that increased protein glycosylation is often 
accompanied by increased phosphorylation of DOL by a CTP- 
dependent kinase; therefore, a second possibility is that DOL 
P is supplied by direct phosphorylation of DOL (Burton et 
al., 1981; Coolbear & Mookerjea, 1981). Third, when the 
sugar residues are transferred to the growing oligosaccharide 
chain, DOL P is released from DOL P-Man or DOL P-Glc. 



20 

Since DOL P is an important precursor of both DOL P- 
monosaccharide and DOL P - o 1 i g o s a c c h a r i d e , it is 
understandable that a shortage of DOL P could have multiple 
effects on the biosynthesis of 1 ip id - o 1 igos acchar i de and may 
cause the production of defective glycoproteins. Chapman has 
shown that a mouse lymphoma cell mutant, lacking DOL P, can 
not synthesize DOL P-Man (Chapman et al . , 1980). Kean 
(1985), using microsomes from a variety of tissues, reported 
that DOL P-Man, which requires DOL P for its biosynthesis, 
exerts a positive allosteric effect on the enzymes that 
catalyze the formation of DOL PP-GlcNac. More recently, 
Carson et al . (1987) found that during hormonal induction of 
glycoprotein assembly in mouse uteri, the changes in the rate 
of DOL P-Man synthesis ' may be an important factor in 
regulating DOL P-linked oligosaccharide assembly, since uteri 
contain very high levels of DOL P and DOL P linked 
saccharides . 

DOL PP, in turn, can arise via two metabolic pathways. 
First, by a recycling mechanism, where DOL PP is released 
from the lipid oligosaccharide when the oligosaccharide 
portion is transferred to the newly synthesized polypeptide. 
Alternatively, DOL PP may be derived from de novo 
biosynthesis by a poorly defined pathway which undoubtedly 
requires the condensation of low molecular weight precursors, 
such as farnesyl diphosphate and isopentenyl diphosphate. 
Regulation of this pathway could be very important for 



21 

controlling DOL P level in the cells, since this pathway is 
the only de novo biosynthesis pathway known and serve as a 
"bridge" connecting the small metabolites, such as acetyl 
CoA, with the large DOL molecules. A key enzyme of this 
pathway is dehydro DOL PP synthase, which catalyzes the 
synthesis of dehydro DOL PP from farnesyl diphosphate and 
isopentenyl diphosphate. Dehydro DOL PP synthase could be an 
important cellular regulator of glycoprotein biosynthesis as 

a consequence of its regulation in the DOL P de novo 

biosynthesis. A postulated pathway showing the important 
role of dehydro DOL PP synthase in DOL P and glycoprotein 
biosynthesis is demonstrated in Figure 1-4. However, this 
enzyme has not been well studied. 

One approach to clarifying the fate of DOL in vivo has 
been to inject this compound into an experimental animal and 
thereafter monitor its appearance in various organs. After 
injection of 3 H-DOL into the bloodstream of a rat, the 
radioactivity rapidly appeared in the high density 
lipoprotein (HDL) fraction of blood, with subsequent uptake 
into all tissues (Keenan et al . , 1977). Since Elmberger 
suggested that rat liver might be the main or exclusive site 
of DOL synthesis, the presence of DOL in the high density 
lipoprotein fraction of blood may point to high density 
lipoprotein as a DOL transporter (Elmberger et al . , 1987). 

The rate of clearance of • L ^C-DOL from tissues of the 
rat is very slow, since about half the radioactivity is 



22 



Isopentenyl Diphosphate 
+ 

Farnesyl Diphosphate 



Dehydro POL PP 
Synthase 



Dehydro DOL PP 



Dehydro DOL P 



Dehydro DOL 



[H2] 



DOL PP 



+[H2] " 



DOL P 
k 



+ [H 2 ] 



GLYCOPROTEIN 
ASSEMBLY CYCLE 
SHOWN IN 

Figure 1 - 3 . 



DOL Kinase 



DOL 



II 



DOL- esters 



Figure 1-4. A putative pathway showing the 

relationship between dehydro DOL PP and glycoprotein 
b iosynthe s i s . 



23 

present in the whole rat and in the liver 24 hr after 
injection. Furthermore, it was still present 20 days later, 
almost entirely as 14 C-D0L. The half-life of DOL P in rat 
liver has been estimated to be 7-12 days on the basis of the 
size of the DOL P pool. The catabolic products derived from 
labeled DOL have not yet been found (Rip et al . , 1985). 

The Importance of Dehvdro DOL PP Synthase 

Studies on developing brain (James & Kandutsch, 1980a), 
and liver (Keller et al . , 1979; Tavares et al . , 1981; Keller, 
1986) have clearly shown that the rates of cholesterol and 
dolichol biosynthesis are regulated independently, although 
they share in part a common biosynthetic pathway. 

Although 3 -hydroxy - 3 -me thylglutaryl CoA reductase (HMG 
CoA reductase), the rate limiting enzyme in cholesterol 
synthesis and whose product, mevalonate , is a metabolic 
precursor of DOL, was shown to be high in these cells, the 
fact that DOL and cholesterol biosynthesis can be 
independently regulated would lead to uncertainty about the 
potential of this enzyme as the regulatory enzyme in DOL 
biosynthetic pathway. 

Dolichol, cholesterol and ubiquinone are all formed 
naturally from mevalonate. During the synthesis, mevalonate 
undergoes a series of reactions and is converted to 
isopentenyl diphosphate, which in turn, is isomerized to form 
dime thylally 1 diphosphate. Then there is a sequential 



24 

condensation of isopentenyl diphosphate first to the 
d im e thy 1 a 1 1 y 1 diphosphate primer and then to the 
consequential allylic diphosphate. Earlier biosynthetic 

experiments confirmed that the isoprene residues of 
polyisoprenoid alcohols are added in a stereochemically 
specific manner (Hemming, 1970). The cis - addition to 
trans . trans - f arnesyl diphosphate gives rise to dolichols, 
and the trans - addition to trans . trans - f arnesyl diphosphate 
generates the polyprenyl side chains of ubiquinones. On the 
other hand, the synthesis of squalene, a cholesterol 
precursor, results from a reductive condensation of two 
molecules of trans . trans - f arnesyl diphosphate (Figure 1-5). 

Many enzymes are common to the pathways of cholesterol 
and DOL biosynthesis. In particular HMG-CoA reductase, which 
is a major regulatory enzyme for cholesterol biosynthesis 
(Faust et al . , 1979; Rodwell et al . , 1976), 

could also control the biosynthesis of isoprene units 
utilized for dolichol synthesis. But, effectors that inhibit 
cholesterol biosynthesis in a major way have minimal effect 
on DOL biosynthesis (James & Kandutsch, 1980b). A current 
debatable hypothesis to explain these findings suggests that 
dehydro DOL PP synthase is saturated at a much lower 
concentration of the prenyl diphosphate substrates than the 
enzymes of cholesterol pathway, and therefore, the synthase 
is less subject to large changes in the availability of these 
substrates (Keller et al . , 1979; James & Kandutsch, 1979). 



25 



Acetyl CoA 



HMG-CoA 



Mevalonate 



reduc tas e 



Isopentenyl Diphosphate 



Farnesyl Diphosphate 



trans-prenyl transferase 




cis-prenyl transferase 



Squalene 



Polyprenyl Diphosphate 
Precursors of Ubiquinone 



Dehydro DOL PP 



Cholesterol 



Dolichol 



Figure 1-5. Pathway of Isoprenoid Biosynthesis. c is - 
prenyl transferase is the same as dehydro DOL PP synthase. 



26 

Therefore, it is likely that the pathway of DOL synthesis may 
have its own regulatory point which is independent of 
cholesterol biosynthesis. This could make dehydro DOL PP 
synthase a r ate - 1 imi t ing step in DOL biosynthesis. This, 
coupled with the finding that DOL synthesis in developing 
systems is greatly enhanced relative to cholesterol 
biosynthesis, makes it likely that large increases in dehydro 
DOL PP synthase activity might accompany or precede an 
increase in glycoprotein synthesis. 

Studies Related to DOL P Biosynthesis and Spermatogenesis 

The Role of Dehydro DOL PP in DOL Metabolism 

Testicular tissues contain large quantities of DOL and 
are actively engaged in glycoprotein synthesis. Early 
studies on human tissues by Rupar and Carroll (1978) using 
gravimetric methods for determining DOL concentrations 
suggested that testis contained more DOL than any other 
organ . 

James and Kandutsch (1980b) suggested that one or more 
of the spermatogenic cell types are responsible for the high 
rate of DOL synthesis observed in normal testicular tissue. 
Further studies of Potter et al. (1981b) showed that purified 
mouse spermatogenic cell populations are capable of DOL 
synthesis. The pachytene spermatocyte were the most active, 
whereas the round spermatids are less active. These studies 



27 

clearly demonstrated high DOL synthesis in specific 
spermatogenic cell populations. However, the enzyme or 
enzymes that might be responsible for this increased DOL 
synthesis were not identified. 

In order to understand how glycoprotein assembly is 
coordinated with differentiation, it is necessary to 
understand how the individual steps in the sequence of 
glycoprotein assembly are modulated. We focus our attention 
here on the biosynthesis of the carbohydrate carrier, DOL PP. 

It is believed that dehydro DOL PP, a derivative of DOL 
PP with an a-unsaturated isoprene unit, is an intermediate in 
the de novo biosynthesis of DOL PP. Figure 1-6 compares the 
structures of dehydro DOL PP and DOL PP. 

The enzyme catalyzing the synthesis of this 
intermediate is dehydro DOL PP synthase. Dehydro DOL PP 
biosynthesis from isopentenyl diphosphate (IPP) and farnesyl 
diphosphate (FPP) has been demonstrated in hen oviduct 
(Grange & Adair, 1977), Ehrlich tumor cells (Adair & 
Trepanier, 1980), chicken liver (Wellner & Lucas, 1979), 
mouse L-1210 cells (Adair & Cafmeyer, 1987a) and yeast (Adair 
& Cafmeyer , 1987b) . 



FPP + (IPP) *► dehydro DOL PP (17-23 x C 5 ) 



28 




Figure 1-6. The structure of dehydro DOL PP and DOL PP. 
The a-unsaturated isoprene unit is present on the structure 
of dehydro DOL PP. 



The condensation of these substrates starts a 
polymerization reaction which finally leads to the formation 
of dehydro DOL PP with farnesyl diphosphate providing the 
three w-terminal isoprene units and the last isopentenyl 
diphosphate added providing the a-unsaturated unit bearing 
the diphosphate. The hen oviduct and Ehrlich tumor cell 
enzymes are membrane associated. 

Dehydro DOL P biosynthesis from isopentenyl diphosphate 
and farnesyl diphosphate has also been described in 
microsomal fractions of rat liver (Wong & Lennarz , 1982). 



29 

Adair and Keller (1982) have showed that the enzymatic 
products of the liver enzyme were a group of dehydro DOL 
monophosphates ranging in size from C75 to C95 (15-19xC5). 
Recent data from this laboratory have shown that rat 
testicular homogenates and their membrane fractions will 
catalyze the synthesis of C75-C85 dehydro DOL P and dehydro 
DOL PP from t,t-farnesyl diphosphate and isopentenyl 
diphosphate (Baba et al . , 1987). The isolation of dehydro 
DOL P and DOL P in some cases instead of the diphosphate 
analogues reflects the activity of diphosphatases . 

A mechanism of reduction of dehydro dolichyl 
derivatives to DOL was suggested recently by Ekstrom et al. 
(1987). They hypothesized that during the saturation of the 
terminal isoprene unit, dehydro DOL PP (also referred to by 
some as polyprenols to distinguish them from the saturated 
counterparts, the dolichols) are elongated by an additional 
isoprene residue and saturated at the same time. 

The enzymes of DOL metabolism are membrane associated 
and, therefore, most often assayed as particulate 
preparations. Solubilization and purification of individual 
enzymes of the dolichol pathway have proven to be difficult 
and in most cases not particularly successful. 



DOL Metabolism in Sp e rma t o gen i c Cells 

James and Kandutsch (1980c*) studied 
X-irradiated or genetically mutated mice 



DOL biosynthesis in 
that were deficient 



30 

in spermatogenic cells. Testes from these mice, when 

compared to normal controls, demonstrated markedly reduced 
ratios of acetate incorporation into DOL as compared to 

cholesterol. These results suggested that the high rate of 
DOL synthesis in mouse testes may be attributed to one or 
more types of spermatogenic cells, although Sertoli cells may 
not be excluded. It was subsequently shown that prepuberal 
mouse pachytene spermatocytes incorporate acetate into DOL at 
a rate which is 5 times higher than that of leptotene and 
zygotene spermatocytes, and that a high rate of acetate 
incorporation into DOL is maintained in adult pachytene 
spermatocytes and round spermatids (Potter et al . , 1981b). 

A developmental study of DOL kinase activity in 
sexually maturing rats, 15-60 days of age, showed the 
appearance of detectable levels of activity at 21 days, a 
peak at 24 days and a subsequent decline to adult levels. 
The developmental pattern of this enzyme suggests its 
association with the later stages of spermatocyte development 
(Berkowitz & Nyquist, 1986). 

Allen and Ward (1987) also reported changes in specific 
activity of DOL kinase during testicular development in the 
rat. They showed that the specific activity of kinase peaked 
at day 30, whereas the level of endogenous DOL P, as measured 
indirectly by the DOL P dependent mannosyl transferase 
activity, rose to a peak around day 15. Since the optimal 
activity of DOL kinase peaked later (day 30) than the peak in 



31 

DOL P levels (day 15), it was suggested that DOL kinase may 
function primarily in maintaining, adequate levels of DOL P 
for glycoprotein biosynthesis after the initial burst of DOL 
P biosynthesis. Therefore, DOL kinase was postulated to have 
little or no effect in regulating the rise in DOL P levels 
during the initial phases of differentiation during 
spermatogenesis. It was suggested instead that dehydro DOL 
PP synthase or DOL PP phosphatase may be the putative 
regulatory enzyme which directly affects the levels of DOL P 
in spermatogenic cells at different stages of development. 
The studies described here will extend our understanding of 
the regulation of DOL P metabolism in the testicular system. 



Glycoproteins in Sertoli Cells 

Sertoli cells are histologically and physiologically 
fundamental for spermatogenesis, since they are the only 
somatic epithelial component of the seminiferous tubules 
(Fawcett, 1975). The close physical association of Sertoli 
cells with the spermatogenic cell and the organization of 
this association into a cyclic pattern have been described in 
detail (Clermont & Perey, 1957). The characterization of 
Sertoli cells as nursing cells of testis was based originally 
on the morphological cellular relationship in the testis. 
The concept of Sertoli cells functioning as a support or 
regulatory factor has been confirmed by both biochemical and 
endocrine studies. It has been postulated that the 



32 

androgenic and tropic hormone action on spermatogenesis is 
mediated by the Sertoli cells. These cells have both FSH and 
androgen receptors (Sanborn et al . , 1977; Sanborn et al., 
1979; Means & Vaitukaitis, 1972) and show an appropriate 
temporal relationship between hormone binding and cell 
response. For example, there is nuclear accumulation of 
androgen and stimulation of RNA polymerase II activity in 
cultured Sertoli cells when FSH or testosterone were added in 
the media (Lamb et al . , 1981). Therefore, it is possible to 
envision a scenario where the regulation of spermatogenesis 
is a result of the biochemical properties of Sertoli cells. 

One of the possible mechanisms for Sertoli cells to 
influence spermatogenic cell development is via the secretion 
of proteins and glycoproteins which serve as signals or 
transport vehicles. The first glycoprotein to be identified 
as a Sertoli cell specific secretion product was androgen 
binding protein (French & Ritzen, 1973; Vernon et al . , 1974). 
This protein is secreted by cultural Sertoli cells and its 
synthesis is regulated by FSH, testosterone, and vitamin A 
(Louis & Fritz, 1977; Karl & Griswold, 1980). The function 
of androgen binding protein is not clear but it is probably 
related to the capability of the protein to bind and 
transport androgens to the epididymis. 

Sertoli cells synthesize and secrete a testicular 
transferrin. Skinner and Griswold (1982) speculate on the 
basis of biochemical and immunological similarities between 



33 

testicular and serum transferrin that testicular transferrin 
must play a role in the transport of iron from Sertoli cells 
to the spermatocytes and spermatids. More recently, Morales 
and Clermont (1986) have shown that during spermatogenesis 
Sertoli cells and spermatogonia internalized transferrin by 
receptor -mediated endocytosis at the base of the seminiferous 
ep i the 1 ium . 

Recent studies in the ram have shown that clusterin, a 
glycoprotein with a molecular weight of 3 7,000-40,000 is also 
synthesized de novo and secreted by Sertoli cells and 
transported to the rete testis (Rosenior et al . , 1987). 

Collectively, Sertoli cells actively secrete 
glycoproteins into the lumen of the seminiferous tubules and 
regulate spermatogenesis. Therefore, it is a relevant 

presumption that the synthesis of glycoproteins must be very 
active in these cells and that adequate DOL P must be 
provided by biosynthesis or uptake to support this function. 
The very high DOL content in Sertoli cell Golgi membrane 
suggests that Sertoli cells are actively involved in 
glycoprotein synthesis (Nyquist & Holt, 1986). 

DOL in Sertoli Cells 

Although a clear relationship between DOL P 
concentrations and the rate of glycoprotein synthesis has 
been established in many systems, the mechanism of regulation 
of glycoprotein biosynthesis in Sertoli cells is still 



34 

obscure. Furthermore, the question concerning the ability of 
the Sertoli cell to synthesize DOL was not addressed 
directly . 

Nyquist and Holt (1986) recently measured the cellular 
and subcellular distribution of DOL in rat testis by a HPLC 
method and found that elutriation purified spermatogenic 
cells had very low concentrations of DOL. Pachytene 
spermatocyte and round spermatids contained 25.8 and 36.5 ng 
DOL/mg protein, respectively. Washed epididymal sperm also 
had a very low DOL content (18.8 ng DOL/mg protein). In 
contrast, the Sertoli cell enriched tubular fraction that was 
recovered during the preparation of purified spermatogenic 
cells showed the highest DOL content (3450 ng DOL/mg 
protein). These results implied that the Sertoli cells 
accumulate a major portion of the testicular DOL. This 
result at first appears inconsistent with the results of 
Potter et al . (1981b), who found low DOL synthesis in the 
testes from X-irradiated mice and spermatogenic deficient 
mice, which, although they were depleted of spermatogenic 
cells, were apparently normal with respect to the number and 
function of the Sertoli cells. Nyquist and Holt suggested in 
explanation that the bulk of testicular DOL may be 
synthesized in the spermatogenic cell and subsequently 
transported to the Sertoli cell. A possible route, they 
hypothesized, would be by Sertoli cell phagocytosis of 
residual body cytoplasm during spermiation. 



35 

The high content of DOL in the Sertoli cell may reflect 
a requirement for high DOL P to permit a rapid rate of 
glycoprotein biosynthesis during the spermatogenesis. It 
might be possible that Sertoli cell and spermatogenic cells 
may have a coordinate pattern of de novo DOL biosynthesis 
which is dependent on the presence of the other cell type. 
This would require that each cell type have the capacity to 
synthesize DOL without relying on intercellular DOL 
transport. In this way the Sertoli cell can independently 
regulate the level of DOL P and hence the synthesis and 
secretion of glycoproteins. Consequently, Sertoli cells can 
regulate spermatogenesis. Therefore, it was of interest to 
determine the capacity of Sertoli cells for de novo 
biosynthesis of DOL P in order to have a better understanding 
of the relationship between the Sertoli cells and 
spermatogenic cells during testicular development. 



S i gni f i c anc e 



Dehydro DOL PP synthase is obviously a prime candidate 
as a regulated enzyme in the DOL biosynthesis and 
glycoprotein synthesis. It was of interest to determine if 
increased dehydro DOL PP synthase activity correlated with 
the increased rate of DOL synthesis in specific types of 
spermatogenic cells and the high DOL content in Sertoli 
cells, particularly since glycoprotein synthesis is active in 



36 

those cells. Recent data from our laboratory using DOL P 
dependent mannosyl transferase show that the level of DOL P 
increases dramatically from day 7 to day 20 in prepuberal 
rats (Allen & Ward, 1987). This time interval covers a 
period when the first group of spermatogenic cells are going 
through differentiation to become spermatids. It should be 
noted that the acrosomal enzymes, including the glycoprotein 
acrosin, are elaborated in early stages of spermatid 
formation. Several other proteins required for glycoprotein 
biosynthesis are also maximally expressed during this time 
interval, e.g. galactosyl transferase, N- acetylglucosaminyl 
transferase, and N- acetylglucosaminide fucosyltransf erase in 
mice testes (Letts et al . , 1974a). It is true for the 
androgen binding protein in rat Sertoli cell culture as well 
(Rich et al . , 1983). Therefore, we have measured the 
specific activity of dehydro DOL PP synthase in spermatogenic 
cells and Sertoli cells during testicular development in 
order to determine its potential importance in the regulation 
of spermatogenesis. 

A knowledge of changes in the concentration of 
intermediates and activities of enzymes in DOL metabolism is 
important if we are to understand the regulation of 
glycoprotein biosynthesis. Spermatogenesis offers a complex 
but good model system to study control mechanisms of DOL 
metabolism and DOL function in the biosynthesis of specific 
glycoproteins during cell differentiation. Information 



37 

gained from the assessment of dehydro DOL PP synthase 
activities in Sertoli cells and different types of 
spermatogenic cells may provide some insight into the 
mechanism of biochemical control of cell function during 
differentiation. This may ultimately be useful in the 
development of male contraceptives. 

Ob j ectives 

The main objectives of this dissertation are to study 
dehydro DOL PP synthase and dehydro DOL PP phosphatase during 
spermatogenesis in rat. 

Specific objectives are the following: 

A. Optimize the assay conditions for dehydro DOL PP 
synthase . 

B. Characterize the enzymatic products. 

C. Determine the synthase specific activities in 
homogenate of testicular tubules of different aged 

rats . 

D. Determine enzymatic activities in homogenate of 
different spermatogenic cell types (pachytene 
spermatocytes and spermatids), and Sertoli cells 

from rats testes. 

Chapter II of this dissertation describes the study in 
achievement of objectives A through C. Chapter III describes 
the work in fulfillment of objective D. 



CHAPTER II 

DEVELOP AND OPTIMIZE AN ASSAY FOR DEHYDRO DOLICHYL 
DIPHOSPHATE SYNTHASE FROM RAT TESTES 



Introduction 

Elucidation of the mechanisms that regulate the 
synthesis of N-linked glycoproteins requires a clear 
understanding of the biosynthesis and metabolism of DOL and 
DOL P. For this reason we have undertaken studies on the 
biosynthesis and metabolism of dehydro DOL PP in rat testes. 
It seems that dehydro DOL PP is the precursor of DOL PP in 
the de novo biosynthesis pathway. Dehydro DOL PP synthase is 
responsible for the de novo biosynthesis of dehydro DOL PP 
from isopentenyl diphosphate and t,t-farnesyl diphosphate. 
This synthase has been demonstrated in several animal tissues 
(Grange & Adair, 1977; Wellner & Lucas, 1979; Wong & Lennarz , 
1982; Adair & Keller, 1982; Adair et al . , 1984; Adair & 
Cafmeyer, 1987a and 1987b; Baba et al . , 1987), and is 
membrane associated. The products of this synthase were 
labile to acid and yielded petroleum ether soluble products 
indicating that the a- isoprene unit was unsaturated. Adair 
and Keller (1982) characterized the carbon number of the 
enzymatic product in rat liver and showed they are a group of 

38 



39 

dehydro DOL Ps ranging in size from C75 to C95. More 
recently, Baba et al . ( 1987 ) described the synthase in rat 
seminiferous tubules, but showed that the products of this 
enzyme were both dehydro DOL PP and dehydro DOL P. 
Hydrolysis of both of these products with a testicular 
phosphatase in the absence of NaF yielded the same chain 
length alcohols (C75.C90). The isolation of 2,3-dehydro DOL 
P and DOL P in some cases instead of the diphosphate 
derivatives undoubtedly reflects the action of a 
diphosphatase . 

The methods for the assay of dehydro DOL PP synthase, 
which were described by Adair and collaborators (Adair & 
Keller, 1982; Adair et al., 1984), and Wellner and Lucas 
(1979) have been modified, as described below, to optimize 
the analysis of synthase activity in whole homogenates of 
small testicular samples obtained at different stages of 
spermatogenesis. In this assay, [ 14 C] -isopentenyl 

diphosphate and t,t-farnesyl diphosphate were chosen as 
substrates. As possible inhibitors of endogenous 

phosphatase, NaF and ATP were used to protect the substrates. 
Although Baba et al . (1987 ) showed that there were no major 
changes in the extent of formation of radiolabeled enzymatic 
products when NaF, MgCl2 and ATP were omitted in the assay of 
partially purified subcellular membrane fraction, there were 
major changes in the nature of the in vitro products. For 
example, when Triton X-100 was omitted neither dehydro DOL PP 



40 

nor dehydro DOL P was formed, but a product, tentatively 
identified as presqualene monophosphate accumulated instead. 
Therefore, it was necessary to extend the earlier studies of 
Baba et al . (1987 ), to ensure that the assay developed for 
the synthase activity in crude tubular homogenates was 
measuring the desired activity. 

This chapter 1) shows that treatment of the testicular 
homogenate by sonication yielded good dehydro DOL PP synthase 
activity while greatly reducing the formation of farnesol via 
another prenyl transferase activity; 2) describes the optimal 
parameters for the dehydro DOL PP synthase assay; 3) 
demonstrates unequivocally the p r e c u r s o r - p r o due t 

relationship between dehydro DOL PP and dehydro DOL P, and 4) 
elucidates a change in the enzymatic activity for dehydro DOL 
PP synthase in the seminiferous tubules during early stages 
of development. A possible role of the dehydro DOL PP 
synthase in regulating the biosynthesis of DOL is discussed. 
The two-fold increase in the specific activity of this 
synthase between day 7 and day 23 and a similar decrease in 
activity between day 23 and day 60 shown in this chapter 
provided the impetus to evaluate (in Chapter III) the enzyme 
specific activity in different cellular populations of rat 
testes . 



41 



Materials and Methods 

Materials . Male Sprague - Dawley rats were purchased 
from local suppliers. t, t-Farnesyl diphosphate was prepared 
as previously described (Baba and Allen, 1978). [1- C]-A - 
Isopentenyl diphosphate and [ 3 2 P ] - or thophosphor ic acid 
(carrier free) in dilute HC1 was purchased from 
Amersham/Searle Corp. Silica gel 60 F254 and Cellulose F254 
on plastic sheets were products of E. Merck. All other 
reagents were obtained from standard commercial sources. 

Solvent Systems. The following solvents were used for 
extraction or chromatography: Solvent A, CHCI3-CH3OH (2:1); 
Solvent B, C H C 1 3 - C H 3 H - H 2 ( 3:4 8:4 7 ); Solvent C, 
diisobutylketone-glacial acetic acid-H20 (8:5:1); Solvent D, 
2 - p r o p ano 1 - a c e t o n i t r i 1 e - . 1 M ammonium bicarbonate 
(45:25:30); Solvent E, 1 - propanol - concentrated ammonia-H20 
(6:3:1) . 

Animal grouping number required for analysis. In order 
to have sufficient data for statistical analysis in each 
experiment, at least two rats, but most often three or more 
rats were used for each age group tested (See Appendix A). 
The excised testes (six or more) from each age group were 
pooled together, then two or three aliquots of the mixed 
samples were taken for assay. The data presented in each 
table or figure were usually means determined by two or three 
similar experiments (e.g. Appendices B and C). 



42 



Preparation of homogenate. 



Male Sprague - Dawley rats 



were decapitated. The testes were removed and perfused with 
enriched Krebs-Ringer bicarbonate medium (EKRB) via the 
testicular vessels. This procedure effectively removed all 
blood cells from testes. The tunica albuginea was removed 
and seminiferous tubules were gently expressed. In the case 
of the younger animals (3 and 7 days old) , the testes and the 
tunica albuginea were removed under dissecting microscope 
without perfusion. 



a ratio of 1:2 (w/v) with ice cold buffer (20 mM Tris-HCl pH 
7.5 and 1 mM EDTA) . This mixture was sonicated in an ice 
bath in a Sonic dismembrator (model 300, Fisher) three times 
for 20 seconds with 20 seconds intervals without sonication. 
The pink milky homogenate was used as the enzyme source. 
Protein quantitation was made by a modified method of Lowry 
et al . (1951). Accurate protein quantitation was extremely 
important for determining enzyme specific activity. 
Therefore, samples were saponified in order to solubilize all 
proteins and each solubilized sample was assayed in duplicate 
or triplicate for protein. 



was developed as adapted from the method described by Grange 
and Adair (1977). The level of the enzyme was monitored by 
measuring the formation of polyprenyl products, dehydro DOL 



The seminiferous 



tubules were weighed then suspended at 



Assay for Dehydro DOL PP Synthase. 



The assay method 



PP and dehydro DOL P. 



It was particularly important to 



43 

eliminate the unwanted products, and only isolate and 
quantitate dehydro DOL PP and dehydro DOL P. Optimal assay 
conditions were also established. The dependency of product 
formation on Triton X-100, protein, isopentenyl diphosphate, 
farnesyl diphosphate concentration and time were determined. 

The standard assay of the enzyme was carried out by 
incubation of 100 mM Tris-HCl buffer pH 7.5, 10 mM MgCl 2 , 
0.5% Triton X-100, 250 /iM t,t-farnesyl diphosphate, 1.6 mM 
ATP, 50 mM NaF, 36 /iM [ 1 - 14 C ]- isopentenyl diphosphate 
(1.1x10^ dpm , 53 /iCi//i mole), and 1.0 mg of enzyme protein in 
a final incubation volume of 0.25 ml for 1 hr at 37°C. The 
reaction was stopped by the addition of 0.25 ml of 1 M KOH . 
Then the mixture was heated at 100° C for 30 min to saponify 
the membrane bound lipids. Afterwards, the mixture was 
cooled in an ice bath, 0.25 ml of 1 M HC1 and 1.25 ml of 2 M 
KC1 were added. This mixture was extracted twice with 1 ml 
aliquots of Solvent A. 

The combined solvent A extract was washed first with 2 
ml of deionized water, then with 2 ml of Solvent B. When the 
extent of product formation was to be quantitated, a 1 ml 
aliquot was taken from the organic extract, dried in a 
scintillation vial and 10 ml of toluene based scintillation 
fluid (Scinti Verse II, Fisher) were added for analysis of 
the radioactivity. Radioactivity was then determined in a 
scintillation counter. 



44 

Thin Layer Chromatography (TLC) of Reaction Products . 

The remainder of extract (4.5 ml) was brought to dryness with 
a N2 stream. Five drops of Solvent A were added to the dried 
residue and the tube was vortexed thoroughly. The resulting 
solution was applied to Silica gel 60 on plastic sheets which 
were previously cut into 4 cm x 20 cm sections. Five 
additional drops of Solvent A were used to wash the sample 
tube and this wash was added to the sample at the origin of 
the TLC sheets. The TLC sheets were then developed in a 
chamber with Solvent C. 

The developed TLC sheets were subjected to either 
scanning with a radiochromatogram scanner (Packard model 
7201) (Fig. 2-1-1) or autoradiography for 3-5 days on X-omat 
AR Kodak film (Fig. 2-1-II). The positions of migration of 
authentic DOL P standard and the radiolabeled products were 
correlated. Sections corresponding to the migration of 

dehydro DOL PP and dehydro DOL P were scraped into 
scintillation vials and 10 ml of scintillation fluid was 
added to each vial for radiochemical analysis. The level of 
activity was expressed in pmoles of [ ^C ] - isopentenyl 
diphosphate incorporated into dehydro DOL PP and dehydro DOL 
P /mg protein. 

Preparation of Isopentenyl fa.i3- 32 P1 Diphosphate. 
Isopentenyl [a,0-**P] diphosphate was synthesized from 3- 
methyl - 3 -buten- 1 - ol and 32 Pi according to the procedure of 
Cramer and Bohm ( 1959 ). [ 3 2 P ] - Or thopho sphor i c acid (0.5 mC^, 



45 

carrier free) in dilute HCl was dried in the reaction vessel 

over P2°5 under N2 . Then 0.5 /imoles crystalline H3PO4, 6.25 

/xmoles triethylamine , 2 pinoles 3 -methyl - 3 -buten- 1 - ol , and 

12 /moles trichloroacetonitr ile in 60 a*1 acetonitrile were 

added and the reaction permitted to proceed for 5-7 hours at 

room temperature. The reaction was stopped by adding 200 fil 

of 10 mM NH4OH. The reaction products was separated by TLC 

(Cellulose F254) in Solvent D. The radioactive ^P-band 

migrating beside authentic [ 14 C ]- isopentenyl diphosphate 

(Rf=0.35) was scraped from the plate, packed in a glass 

column and [ 32 P ]- isopentenyl diphosphate was eluted with 2« ml 

3 2 

of methanol at room temperature. The nature of the P- 
labeled product was verified by TLC along with authentic 
isopentenyl diphosphate by TLC on Silica 60 F254 in Solvent 
E . 

Base Hydrolysis of Dehvdro Pol PP. The putative 
dehydro DOL PP was biosynthes ized from [ 14 C ] - i s openteny 1 
diphosphate and farnesyl diphosphate with a testicular 
homogenate according to the standard assay method. Enzymatic 
products were separated by TLC on Silica 60 F254 as already 
described. The dehydro Dol PP region on TLC (Rf-0.40) was 
localized by autoradiography and scraped into a conical test 
tube. The product bound to Silica gel was suspended in 1 ml 
of 3.5 M KOH in 70% methanol and the hydrolysis was carried 
out at 100° C for 2 hr . At the end of the hydrolysis, 2 ml 
of water was added to the mixture and the polyprenol 



46 

hydrolysis products were extracted with 2 ml of Solvent A. 
Then the lower phase was subjected to TLC analysis. The 
authentic markers, [ ^C ] - dehydr o DOL P and non- radiolabeled 
DOL P were chromatographed in parallel to the hydrolysis 
products and the developed TLC sheet was subjected to 
autoradiography. 



Results 



Optimization of Synthase Reaction and Characterization 
of the Enzymatic Products . The typical prenyl transferase 
assay for the long chain polyprenyl diphosphate synthase in 
bacteria measures the amount of acid labile and organic 
solvent extractable product (Keenan & Allen, 1974). However, 
this method could not be satisfactorily applied for the 
quantitation of dehydro DOL PP synthase, because of the 
synthesis of other isoprenoid products with acid lability and 
extractablity similar to the dehydro DOL phosphates. A more 
accurate method was developed which involved 1) CHCI3-CH3OH 
extraction of the reaction products after saponification of 
the reaction components, 2) application of TLC to separate 
the dehydro DOL PP and dehydro DOL P from shorter chain 
polyprenyl phosphates and free polyprenols, and 3) 
determination of the sum of the dehydro DOL PP and dehydro 
DOL P formed. This method was satisfactorily applied to the 
identification of dehydro DOL PP synthase in the microsomal 



47 

fraction isolated from homogenates of seminiferous tubules 
from rat testes (Baba et al . , 1987). 

It was necessary to optimize the method to accurately 
assay the enzyme in homogenates of tubules taken from animals 
of different ages. Homogenates were prepared by the 

sonication of buffered suspensions of tissue instead of 
disruption with a glass homogenizer as previous described 
(Baba et al . , 1987). In this study, sonication was found to 
be the only satisfactory procedure for disruption of the 
small amounts of tissue available from 3- and 7-day-old rats. 
Sonication also had the added advantage of denaturing the 
prenyl transferase, farnesyl diphosphate synthase, as 
exhibited by an elimination of radioactive product 
chromatographing with an Rf similar to exogenously added 
farnesol (Fig. 2-1-1, Panel B). 

•*2 p and Ratios in the Mono and Diphosphate 

Products . The carbon chain lengths of the long chain 
polyprenyl products obtained by in vitro biosynthesis were 
previously shown to be the same (Baba et al . , 1987). Since 
these two compounds had the same chain length, but they 
chromatographed on TLC and anionic exchange columns in a 
manner consistent with mono- and diphosphorylated products, 
they were assumed to be the dehydro Dol P and dehydro Dol PP. 
The ratios of phosphate to polyprenol chain were determined 
here for the putative dehydro Dol P and dehydro Dol PP in 
order to clarify this earlier assumption. Mixtures of [a,/i- 



48 

3 2 P ] - i s o p e n t e ny 1 diphosphate and [ 14 C] -isopentenyl 
diphosphate were incubated with farnesyl diphosphate and 
tubular homogenates in the standard assay. The products were 
separated by TLC as usual and the ratios of radiolabel 
incorporated from [ 32 P]- and [ ]- isopentenyl diphosphate 
were determined. Since the chain lengths of the polyprenol 
products have been established to be the same, the relative 
ratio of 32 P incorporated/^C incorporated into the 
polyprenyl diphosphate is expected to be twice that ratio for 
the monophosphate. The results of such a test under two 
experimental incubation conditions are illustrated in Table 
2-1. They support the hypothesis that the two products are 
the dehydro DOL P and dehydro DOL PP. 

Several experiments were carried out to demonstrate the 
dependency of the enzyme activity on Triton X-100 
concentrations and incubation times as well the extent and 
type of product formed. These experiments also serve to test 
the p recur s or - produc t relationship between the diphosphate 
and monophosphate . 

Triton X-100 stimulated dehydro DOL PP synthase 
activity (Fig. 2-2, Panel C) with formation of dehydro DOL PP 
(Panel A) and dehydro DOL P (Panel B). The dependencies of 
the extent of mono- and diphosphate product formation on the 
concentration of detergent were quite different after 
incubation for 1 hr . Dehydro DOL PP formation was generally 



Figure 2-1. 



Separation of 



Enzymatic 



Products by TLC . 



(I) The products from the reaction of t , t-farnesyl 
diphosphate and [ ^ ] - i s openteny 1 diphosphate with 
homogenates of seminiferous tubules prepared with a 
glass-teflon homogenizer (Panel A) or by sonication 
(Panel B) were extracted with CHCI3/CH3OH and subjected 
to TLC on silica gel sheets as described in the text. 
Arrows represent the position of migration of exogenous 
DOL P and f arnesol . 



(II) A example of autoradiography from enzyme assay. 



50 








J i- 

5 10 

DISTANCE (cm) 



J 

15 SF 



SF - 



DOLP 




Figure 2-1 (continued) 



Table 2-1 



Incorporation of A-^-fl-^CI Isopentenvl Diphosphate and f a . g-^ - PI -Isopentenvl 
Diphosphate into Dehydro POL PP and Dehvdro POL P a 



Radiolabeled 
Substrate 



Experiment 1 



[32pj _ Ipp 

5 nmol 
(1.36 jiCi) 



[ 14 C]-IPP 
4 nmol 
(0.21 jiCi) 



[ 32 P] -IPP 



Experiment 2 

[ 14 C]-IPP 
4 nmol 



RATIO 

32 Pcpm 10 nmol 
i4 Ccpm (2.72 /id) (0.21 ^Ci) 



RATIO 

32 Pcpm 

1 *Ccpm 



Radiolabeled 
Product 

Dehydro DOL PP (A) 



(cpm incorporated) 



424 



1489 



0.28 



(cpm incorporated) 
1127 883 



1.28 



Dehydro DOL P (B) 



217 



1526 



0.14 



533 



936 



0.57 



(A)/(B) 



2.0 



2.2 



a The enzyme assay was carried out as described in the text. The reported values of 
cpm have been corrected for overlap of 32 P into the channel. (A)/(B) represents 

32 P incorporated into the diphosphate compared 



the relative ratio of radiolabel from 
to the monophosphate . 



Figure 2-2. Triton X-100 Dependency on the Formation of 
Dehydro DOL PP and Dehydro DOL P. 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl2, the indicated percentage of Triton 
X-100, 250 /iM t,t-farnesyl diphosphate, 1.6 mM ATP, 50 
mM NaF, 36 /iM [ 1 - ] - isopentenyl diphosphate, and 1.0 
mg of enzyme protein in a final volume of 0.25 ml were 
carried out at 37° C for 60 minutes. The formation of 
[ 14 C] -dehydro DOL PP (Panel A) and [ 14 C ]- dehydro DOL P 
(Panel B) were estimated by the method described before. 
Panel C represents dehydro DOL PP synthase activity 
(A+B) . 



54 




55 

stimulated with increasing detergent concentration throughout 
the concentration range shown in the figure, while dehydro 
DOL P formation was optimal at 0.5% Triton X-100. This 
suggests that when the Triton X-100 concentration was higher 
than 0.5%, a previously active dehydro DOL PP diphosphatase 
was inhibited. The sum of dehydro DOL PP and dehydro DOL P 
production was unchanged at Triton X-100 concentrations of 
0.5% and higher . 

The product ratio of dehydro DOL P to dehydro DOL PP 
shifted to favor the monophosphate when the detergent 
concentration in the incubation mixture was decreased midway 
through the incubation period. A part of the tubular 
homogenate was incubated at 37° C with substrates in either 
0.5% Triton X-100 or 2% Triton X-100. In each case the 
products were analyzed after 1 hr and 2 hr . The incubation 
with 0.5% Triton X-100 gave dehydro DOL P as the predominant 
product at both 1 hr and 2 hr (Fig. 2-3, Panel A), whereas 
with 2% Triton X-100, the slower migrating product, dehydro 
DOL PP, was the predominant product at both time points (Fig. 
2-3, Panel B) . However, when a similar incubation was 
carried out in 2% Triton X-100 for the first hour to favor 
dehydro DOL PP formation and then the concentration of Triton 
X-100 changed to 0.5% during the second hour of incubation, 
the predominant product observed at the end of the second 
hour was dehydro DOL P (Fig. 2-3, Panel C). 



Figure 2-3. Dependence of Product Formation on Triton 
X-100 Concentration and Incubation Time. 

Sonicates of seminiferous tubules were assayed 
under standard conditions except tbat Triton X-100 and 
time of incubation were varied as shown. Dehydro DOL PP 
and dehydro DOL P were analyzed separately in reaction 
mixtures incubated for 1 hour and 2 hours in 0.5% Triton 
X-100 (Panel A), 2% Triton X-100 (Panel B) and from a 
reaction mixture which was incubated for 1 hour in 2% 
Triton X-100 and then diluted four fold with all 
reaction constituents except enzyme and Triton X-100 and 
incubated for an addition 1 hour (Panel C). 



1st hour 



2nd hour 



1 

0.5%Triton X-100 



A. 





2%Trtton X-100 



B. 




2%Trtton X-100 





0.5%Triton X-100 



C. 




Dehydro Dehydro Dehydro Dehydro 
DoIPP DolP DoIPP DolP 



58 



Table 2-2 

Formation of Enzymatic Product at Different Triton X-100 
Concentrations in Pulse-Chase Experiment 3 



Isopentenyl Diphosphate Incorporated 
(pmoles) 

Time Conditions dehydro DOL PP % dehydro DOL P % 

1st hour 2% Triton 68 71 27 29 

2nd hour 0.5% Triton 49 41 70 59 



a Incubation conditions were the same as described in legend of Fig. 2-3 
except that 4.81 mM unlabeled isopentenyl diphosphate was added to the 
reaction mixture after the first hour, reducing the specific activity of 
[ C] - isopentenyl diphosphate 134-fold. 



59 

In all cases the sum of the two products increased with 
increasing time. This supports the presence of a 

diphosphatase which is inhibited by higher concentrations of 
Triton X-100. 

The results of a similarly designed pulse-chase 
experiment support the same conclusion (Table 2-2). In this 
case non- radiolabeled isopentenyl diphosphate was added to 
the incubation mixture after one hour incubation in 2% Triton 
X-100. The reaction was continued for an another hour with 
addition of farnesyl diphosphate and enzyme but under lower 
Triton X-100 concentration (0.5%). Any polyprenyl phosphate 
made during the second hour of incubation, the chase phase, 
would not have been radiochemically detectable under the 
conditions used. Since there was a loss in radiolabeled 
product migrating as the putative dehydro DOL PP concomitant 
with an increase in radiolabeled dehydro DOL P, it can be 
concluded that the slower migrating product is a precursor of 
dehydro DOL P, and therefore it was dehydro DOL PP. 

The precursor-product relationship between dehydro DOL 
PP and dehydro DOL P was also shown in a more detailed time 
dependent study illustrated in Fig. 2-4. The formation of 
the mono- and diphosphate was determined under standard assay 
conditions except that Triton X-100 concentration was raised 
to 1% to partially inhibit the diphosphatase activity. The 
distinct lag in the formation of dehydro DOL P relative to 
the diphosphate illustrates clearly the classical precursor- 



60 

product pattern. The formation of the two products increased 
linearly with time up to 60 min. 

Base Hydrolysis of Dehvdro POL PP . The putative 

dehydro DOL PP was isolated by TLC and then subjected to 
saponification in 3.5 M KOH in 70% methanol for 2 hr at 
100° C. Figure 2-5 shows that dehydro DOL P is the 

overwhelming product of base hydrolysis. This product is 
consistent with this enzymatic product being dehydro DOL PP. 

Saponification in aqueous 0.1 M KOH at 100° C for 30 
min is used in the standard assay. This procedure makes 
nonsaponif iable lipids more accessible for extraction and 
provides a better resolution of the enzymatic products on 
TLC, because of the hydrolysis and hence elimination of 
saponifiable lipids. Saponification under these milder 

conditions did not change the ratio between dehydro DOL PP 
and dehydro DOL P (results are not shown). 

These results indubitably showed the in vitro reaction 
proceeds as follows: dehydro DOL PP synthase catalyzes the 
condensation of isopentenyl diphosphate and farnesyl 
diphosphate to generate dehydro DOL PP, which is the 
precursor of dehydro DOL P. Since the proportion of the 
diphosphate and monophosphate occasionally varied from one 
experiment to another, an accurate measurement of synthase 
activity required analysis of both products. 



Figure 2-4. Time Course of Dehydro DOL PP and Dehydro 
DOL P Formation. 

Sonicated seminiferous tubules were assayed under 
standard conditions except that 1.0% Triton X-100 was 
used and the time of incubation was varied as shown. 
The formation of dehydro DOL PP (solid triangles), 
dehydro DOL P (solid squares) and the sum of the both 
(solid circles), respectively. 




TIME (min) 



Figure 2-5. Product of Base Hydrolysis of Dehydro DOL 
PP . 

Dehydro DOL PP and dehydro DOL P were prepared by 
biosynthesis and isolated by TLC as described in the 
Methods. (A) Dehydro DOL PP was saponified. The 

hydrolysis product was extracted and chromatographed on 
Silica 60 F254 in Solvent C as described in the text. 
(B) as a control, dehydro DOL P was extracted from 
Silica 60 TLC sheets and chromatographed as described in 
A. The position of migration of authentic DOL P is 
shown by the arrow I. The position of dehydro DOL PP is 
shown by the arrow II. 



64 




- 

A 



B 



65 

Kinetics of the Enzyme . The enzyme assay conditions 
were optimized. Figure 2-6 shows the effect of varying 
protein concentration on the formation of the dehydro DOL PP 
(Panel A), dehydro DOL P (Panel B) and the sum of these two 
products (Panel C). These results indicated that the 

enzymatic activity increased linearly with protein 
concentration up to 2.4 mg protein incubated. 

The dependency of dehydro DOL PP and dehydro DOL P 
formation on substrate concentration was studied. Figure 2-7 
shows that the formation of dehydro DOL PP (Panel A), dehydro 
DOL P (Panel B), and the sum of these two products (Panel C) 
increased with increasing isopentenyl diphosphate 
concentration. A double - rec iprocal plot of the sum of 
dehydro DOL PP and dehydro DOL P formation (Panel C) versus 
isopentenyl diphosphate concentrations showed (Figure 2-8) 
that the apparent Km-32/xM and Vmax-1.23 pmoles/mg protein/min 
respectively . 

Similarly, the dependency of the enzymatic products 
formation on the substrate farnesyl diphosphate was also 
studied. The experiment described (Figure 2-9) shows that 
farnesyl diphosphate was incorporated into dehydro DOL PP and 
dehydro DOL P. The formation of dehydro DOL PP (Panel A), 
dehydro DOL P (Panel B), and the sum of these two products 
(Panel C) increased with increasing concentration of farnesyl 
diphosphate. In Figure 2-10, a double - reciprocal plot of the 
sum of dehydro DOL PP and dehydro DOL P formation (Fig. 2-9, 



66 

Panel C) versus farnesyl diphosphate concentrations showed 
that the apparent Km=22 2/iM and Vmax = 0.67 pmoles/mg 
protein/min respectively. The Km values were higher than 
those observed for dehydro DOL PP synthase from Ehrlich 
ascites (Adair et al . , 1984). This may reflect non-specific 
absorption of the substrates by other proteins in the crude 
homogenate and hydrolysis of the substrate by endogenous 
phosphatases, although ATP and NaF were included in the assay 
to minimize the action of phosphatases. 

The time course of formation of dehydro DOL PP and 
dehydro DOL P in the standard assay is shown in Fig. 2-11. 
The formations of both dehydro DOL PP (Panel A) and dehydro 
DOL P (Panel B) are increased with increasing incubation 
time. Since the sum of these two products was linearly 
increased up to 60 minutes (Panel C), we chose one hour as 
the standard incubation time. 

Changes of Dehydro DOL PP Synthase with age . The 
specific activity of dehydro DOL PP synthase in testicular 
homogenates of different aged rats was studied. The 
synthesis of dehydro DOL PP and dehydro DOL P from farnesyl 
diphosphate and [ ] - isopentenyl diphosphate was determined 
in sonicates of tubules from rats aged 3-65 days. An example 
from several of these experiments is shown in Fig. 2-12. 
Under standard assay condition, the changes in the specific 
activity of the enzyme as measured by dehydro DOL PP 



Figure 2-6. Effect of Protein Concentration on Dehydro 
DOL PP and Dehydro DOL P formation. 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl 2 , 0.5% Triton X-100, 250 fxK t,t- 
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF , 36 fxR [1- 
^C] -isopentenyl diphosphate, and varying protein 
concentration in a final volume of 0.25 ml was carried 
out at 37° C for 60 minutes. The formation of [ 14 C]- 
dehydro DOL PP (Panel A) and [ 14 C ]- dehydro DOL P (Panel 
B) were estimated by the method described before. Panel 
C represents dehydro DOL PP synthase activity (A+B) . 



68 




Figure 2-7. Isopentenyl Diphosphate Concentration 

Dependency on the Formation of Dehydro DOL PP and 
Dehydro DOL P. 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl 2> 0.5% Triton X- 100, 250 /*M t,t- 
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF , the 
indicated concentration of [ 1 - ] - isopentenyl 
diphosphate, and 1.0 mg of enzyme protein in a total 
volume of 0.25 ml were carried out at 37° C for 60 
minutes. The formation of [ 14 C ]- dehydro DOL PP (Panel 
A) and [ 14 C ]- dehydro DOL P (Panel B) were estimated by 
the method described before. Panel C represents dehydro 
DOL PP synthase activity (A+B) . 



70 




IPP CuM) 



Figure 2-8. A double reciprocal plot of the sum of 
dehydro DOL PP and dehydro DOL P formation (Fig. 2-7. 
Panel C) vs. isopentenyl diphosphate concentrations is 
presented. 



72 




Figure 2-9. Farnesyl Diphosphate Concentration 

Dependency on the Formation of Dehydro DOL PP and 
Dehydro DOL P. 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl2 , 0.5% Triton X-100, varying 
concentrations of t,t-farnesyl diphosphate, 1.6 mM ATP, 
50 mM NaF, 36 [ 1 - 14 C ] - isopentenyl diphosphate, and 

1.0 mg of enzyme protein in a final volume of 0.25 ml 
were carried out at 37° C for 60 minutes. The formation 
of [ 14 C] -dehydro DOL PP (Panel A) and [ 14 C ]- dehydro DOL 
P (Panel B) were estimated by the method described 
before. Panel C represents dehydro DOL PP synthase 
activity (A+B) . 




FPP (uM) 



Figure 2-10. A double reciprocal plot of the sum 
of dehydro DOL PP and dehydro DOL P formation (Fig. 2-9. 
Panel C) vs. farnesyl diphosphate concentration is 
presented . 



76 




Figure 2-11. Time Course of Incorporation of [ C]- 
Isopentenyl Diphosphate into Dehydro DOL PP and Dehydro 
DOL P. 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl 2 , 0.5% Triton X- 100, 250 (M t,t- 
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF , 36 pM [1- 
] - isopentenyl diphosphate, and 1.0 mg of sonicated of 
seminiferous tubules as enzyme protein in a total volume 
of 0.25 ml were carried out at 37° C for the indicated 
times. The formation of [ 14 C ]- dehydro DOL PP (Panel A) 
and [ 14 C ]- dehydro DOL P (Panel B) were estimated by the 
method described before. Panel C represents dehydro DOL 
PP synthase activity (A+B) . 



78 




Figure 2-12. Dehydro DOL PP Synthase in Testicular 
Hotnogenate of Different Aged Rats. 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl 2 , 0.5% Triton X- 100, 250 t,t- 
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, 36 ^M [1- 
l^C ] - isopenteny 1 diphosphate, and 1.0 mg of enzyme 
protein from indicated aged rats in a final volume of 
0.25 ml were carried out at 37° C for 60 minutes. The 
formation of [ 14 C ]- dehydro DOL PP (Panel A) and [ 14 C]- 
dehydro DOL P (Panel B) were estimated by the method 
described before. Panel C represents dehydro DOL PP 
synthase activity (A+B). 




DAYS AFTER BIRTH 



Figure 2-13. Dehydro DOL PP Synthase Activity in 
Sonicates of Tubules from Rats of Different Ages. 

The enzymatic activity was assayed under standard 
conditions with sonicates of seminiferous tubules as 
described in the Methods. The data presented as the 
mean + standard deviation (x + "7^= ) • Numbers in 
parentheses indicate the number of animals used to 
prepare the tubules. 



82 




83 

formation (Panel A) were somewhat smaller than changes in 
dehydro DOL P formation (Panel B) during testicular 
development. The sum of dehydro DOL PP and dehydro DOL P 
(Panel C) represents total specific activity of this enzyme. 
A composite of data from these studies using 118 rats is 
shown in Fig. 2-13. A two fold increase in tubular activity 
of the synthase occurred between day 7 and day 23 and a 
similar decrease in activity occurred between day 23 and day 
60. A statistical treatment (Wilcoxon two-sample rank test) 
(Schefler, 1984) of these data shows a significant difference 
between 1) the 7-day-old and the-15-day old groups of rats 
(p<0.005) and, 2) the 30-day-old and the 60-day-old group of 
rats (p<0.01). Therefore, the peak of activity at 23 day old 
rats must be significantly higher than activity in rats aged 
both 7 days and 60 days. 

Discussion 

Early in vivo experiments have demonstrated that 
mevalonic acid serves as a precursor of dolichol in pig, 
rabbit and rat 1 iver (Butterworth et al . , 1966j Martin & 
Th orne, 1974). Using doubly and stereospecifically 

radiolabeled mevalonate and tissue slices, it was also shown 
that dolichol was synthesized from all - trans -farnesyl 
diphosphate by cis - addition of isoprene units (Gough & 
Hemming, 1970), suggesting that the biosynthetic pathway to 



84 

DOL branches from that to cholesterol at the level of 
farnesyl diphosphate. In vitro experiments with isopentenyl 
diphosphate as a precursor have shown that 2,3-dehydro DOL P, 
presumably one of the later intermediates in the DOL 
biosynthetic pathway, could be synthesized in preparations 
from hen oviduct (Grange & Adair, 1977), avian liver (Wellner 
& Lucas, 1979), Ehrlich tumor cells (Adair & Trepanier, 
1980), mouse L-1210 cells (Adair & Cafmeyer, 1987a), yeast 
(Adair & Cafmeyer, 1987b), and testes (Baba et al . , 1987). 
Testis has been shown to contain large quantities of dolichol 
(Rupar & Carroll, 1978; Tollbom & Dallner, 1986; James & 
Kandutsch, 1980c) , and therefore it seems to be an 
appropriate tissue in which to investigate DOL biosynthesis. 

Homogenates of rat seminiferous tubules have been 
previously shown to catalyze the synthesis of acid labile 
polyprenyl mono- and diphosphate (the a- unsaturated isoprene 
unit is acid labile). The enzymatic activity was dependent 
upon t,t-farnesyl diphosphate, isopentenyl diphosphate and 
divalent cation (Baba et al., 1987). 

The conditions for the assay of dehydro DOL PP synthase 
in sonicates of rat seminiferous tubules have been 
systematically characterized and optimized here. The 
sonication of seminiferous tubules gives a few advantages for 
the assay. The sonication method, in contrast to 

homogenization with a glass homogenizer, can be easily 
applied to a small amount of testicular tissue, such as 



85 

obtained from 3- and 7-day-old rats. For instance, the 

pooled size of ten testes from 3-day-old rats is about the 
size of a rice grain (not a long grain!). Furthermore, 
sonication denatures the prenyl transferase, farnesyl 
diphosphate synthase, so that some of the side products from 
the assay are eliminated (Fig. 2-1-1). The reason for the 
loss in this prenyl transferase activity on sonication is not 
clear. Possibly, these enzymes are more sensitive to the 
heat generated by the sonication, despite cooling during this 
process . 

It was necessary to optimize the conditions to 
accurately assay the specific enzymatic activity in sonicated 
seminiferous tissue from rats of different ages. Several 
pieces of evidence support the premise that the slow 
migrating TLC component (Rf=0.47) (Fig. 2-1), identified as 
dehydro DOL PP, was the initial enzymatic product, which was 
subsequently hydrolyzed in vitro to dehydro DOL P. Earlier 
work showed that the chain length of both the mono- and 
diphosphate derivatives of the polyprenyl product were the 
same (C75-C90) (Baba et al . , 1987). The present study 
established more clearly the p r e cur s o r - pr oduc t relationship 
between the products dehydro DOL PP and dehydro DOL P. 
First, the sum of the mono- and diphosphorylated polyprenols 
increased linearly with a variety of increasing variables, 
i. e. Triton X-100 concentration (0 to 0.5% as shown in Fig. 
2-2), protein (0 to 2.4 mg as shown in Fig. 2-6), time (0 to 



86 

60 min as shown in Fig. 2-11). Second, there is evidence for 
a detergent sensitive phosphatase that acts on the 
diphosphate to give the monophosphate. Third, kinetic 

experiments have shown a classical time dependent lag in 
monophosphate formation compared to diphosphate formation, 
whereas total phosphorylated polyprenol increased linearly. 
Fourth, direct chemical experiments were also performed to 
establish the pr e cur s o r - pr oduc t relationship between dehydro 
DOL PP and dehydro DOL P, such as double labeling experiment 
using [ a,p- * * P ] - i s openteny 1 diphosphate and [ ] - isopentenyl 
diphosphate (Table 2-1), and base hydrolysis (Fig. 2-5). 

The mild saponification used in each assay to release 
the membrane bound lipids did not change the ratio between 
dehydro DOL PP and dehydro DOL P. However, during a more 
vigorous saponification, dehydro DOL PP was hydrolyzed to 
dehydro DOL P as previous described (Adair and Cafmeyer, 
(1987). These experiments clearly showed that we can simply 
sum the radioactivity in the mono and diphosphate components 
as an accurate measurement of the total synthase activity. 

Grange and Adair (1977) observed and therefore measured 
only dehydro DOL P formation in hen oviduct, although they 
commented that dehydro DOL P may be derived from dehydro DOL 
PP by the action of a phosphatase. In contrast, Adair and 
Cafmeyer (1987b) observed and measured only dehydro DOL PP 
formation in their studies in yeast. The current study 
showed that the testicular enzyme produces both dehydro DOL 



87 

P and dehydro DOL PP , therefore, the determination of 
synthase activity required the measurement of both products, 
not dehydro DOL P or dehydro DOL PP alone. 

DOL P is an indispens ible carrier of oligosaccharides 
during glycoprotein biosynthesis, therefore, knowledge of its 
availability and the timing of its biosynthesis during early 
stages of differentiation may be important in understanding 
the regulation of spermatogenesis. This laboratory has shown 
that DOL P, as measured indirectly by a DOL P dependent 
mannosyl transferase assay, increased in immature rat testes 
about two fold between day 7 and day 30 after birth (Allen & 
Ward, 1987). Unpublished work of others in this laboratory 
has demonstrated similar results by direct measurement of DOL 
P with HPLC analysis of CHCI3/CH3OH extracts of seminiferous 
tubules from different aged rats (Fig. 2-14). 

Changes in dehydro DOL PP synthase during 
spermatogenesis in the rat have been studied here. The 
products of this synthase are undoubtedly intermediates in 
DOL P(P) biosynthesis. Dehydro DOL PP synthase was shown to 
increase two fold in specific activity between day 7 and day 
23 after birth, and a similar decrease in activity between 
the day 23 and day 60. These findings parallel the changes 
in DOL P described above. Mechanisms which account for the 
increase in DOL P may include the phosphorylation of DOL with 
CTP dependent DOL kinase, de novo biosynthesis of DOL or as a 
result of release of DOL P from pools of DOL P and DOL PP 



Figure 2-14. Comparison of Changes in DOL P Concentration, 
and Dehydro DOL PP Synthase Activity as a Function of Rat 
Age . 

The specific activity of dehydro DOL PP synthase ( ) 

are compared with the concentration of DOL P measured 
directly by HPLC (A) (unpublished observation, Allen, 1987) 
or by an indirect method (□), which was described in an 
earlier study (Allen & Ward, 1987). 



89 




DAYS, AGE 



90 

saccharides. Berkowitz and Nyguist (1986) showed a sharp 
rise in kinase activity at 21 days of age with a peak at 24 
days. Allen and Ward (1987) showed a similar change in 
kinase activity, but the rise in activity appeared to be more 
gradual with a peak in activity at about 30 days and with one 
half maximal activity between day 20 and day 25. It seems 
unlikely that change in DOL kinase activity account for the 
changes in DOL P levels. 

It has been suggested that alterations in the levels of 
the active, phosphorylated form of dolichol regulate the rate 
of N-linked glycoprotein synthesis (Lucas, 1979; Carson & 
Lennarz , 1979; Carson & Lennarz, 1981). The present results, 
which show that dehydro DOL PP synthase changes in specific 
activity at early stages of testicular development in rats 
(Fig. 2-13) suggest that increased DOL levels must accompany 
fluctuations in glycoprotein biosynthesis observed during 
this time period (Letts et al., 1978). The high rate of 
testicular DOL synthesis shown by Kandutsch and co-workers, 
as well as the temporal changes in DOL metabolism shown here 
during sperm differentiation, strongly suggest that membrane 
glycoproteins and alterations in the timing of their 
appearance may be significant regulators of spermatogenesis. 

Potter et al . (1981b) showed a high rate of acetate 
incorporation into DOL in pachytene spermatocytes of adult 
mouse testes. At the same time, they also found increased 
HMG CoA reductase activity in these cells. Although HMG CoA 



91 

reductase may be one of the regulated enzymes in DOL 
synthesis (Rodwell et al . , 1976; James & Kandutsch, 1979), 
the observed independent regulation of DOL and cholesterol 
biosynthesis can not be well explained by the control of this 
enzyme alone. A strong case is presented here for a 
regulatory role for dehydro DOL PP synthase during de novo 
DOL biosynthesis, because the specific activity of this 
enzyme rises in parallel with a two to three fold increase in 
DOL P concentration measured both directly and indirectly. 
Therefore, it can be concluded that dehydro DOL PP synthase 
is a regulatory enzyme responsible for controlling DOL 
biosynthesis on a pathway that is independent of cholesterol 
biosynthesis. This conclusion is consistent with the 

findings of Keller and Adair (1980), that DOL P synthase or 
long chain cis -prenyl transferase is a rate limiting factor 
in the biosynthesis of DOL P in liver. Recently, when 
radioactively labeled mevalonate was utilized to study in 
vivo and in vitro cholesterol, DOL and ubiquinone 
biosynthesis, considerable differences were observed between 
the rate of cholesterol synthesis and the rate of DOL and 
ubiquinone synthesis, while the rates of DOL and ubiquinone 
synthesis were quite similar (Elmberger et al . , 1987). This 
observation suggested that the presence of important rate- 
limiting steps in the biosynthesis of DOL and cholesterol 
after mevalonate. This study suggests that dehydro DOL PP 
synthase may be one of these rate - limiting factors. 



92 

Since the cellular composition of the seminiferous 
tubules differs as a function of age during early stages of 
differentiation, changing cell populations may partially 
explain the difference in activity of dehydro DOL PP synthase 
in testes from rats of different ages. For instance, in 7- 
day-old rats the only spermatogenic cells are spermatogonia, 
by day 23, there are spermatogonia and pachytene 
spermatocytes as well, and after day 26, spermiogenic cells 
start appearing in the seminiferous tubules. It is likely 
that the different enzymatic activity observed during 
development can be accounted for by different cell 
populations with different enzymatic activity. The specific 
activity of different cellular populations are elaborated in 
the next chapter. 



CHAPTER III 

DEHYDRO DOLICHYL DIPHOSPHATE SYNTHASE ACTIVITY IN 
ENRICHED CELL POPULATIONS FROM RAT TESTIS 



Introduction 

Although DOL metabolism during testicular development 
has been the subject of several studies in recent years, a 
number of intriguing questions still remain unanswered. For 
example, do all rat spermatogenic cell populations have 
dehydro DOL PP synthase activity? Does each subpopulation of 
spermatogenic cells have the same enzymatic activity for the 
synthase? Is there a temporal relationship between synthase 
activity and developmental stage? Do Sertoli cells also have 
the synthase activity and synthesize its own dolichol? Is 
there a temporal relationship between spermatogenic cell and 
Sertoli cell synthase activity? Elucidating the answers to 
these questions will be of value in understanding the 
regulation in DOL metabolism as well as glycoprotein 
biosynthesis during spermatogenesis in rat. In this chapter, 
the specific activity of 2,3-dehydro DOL PP synthase in 
homogenates of protease treated seminiferous tubules, cell 
fractions enriched in spermatogenic cells or Sertoli cells 
from testis were measured as a function of the age of 

93 



94 

prepuberal rats. The highest activity of this enzyme 

occurred in each case with cells from rats aged 23 days. 
Homogenates of cell fractions enriched in pachytene 
spermatocytes, spermatids or Sertoli cells were found to have 
higher synthase activity than a whole testicular homogenate 
or a mixture of cells prepared by protease treatment of 
tubules. The specific enzymatic activity in pachytene 

spermatocytes expressed per mg protein, was about 1.7 fold 
higher than in spermatids and about 8.3 fold higher than in 
spermatozoa. Therefore, the increase in spermatogenic cell 
synthase before day 23 can be accounted for by the appearance 
of the pachytene spermatocytes. Generally speaking, little 
net increase in enzyme occurred during or after meiotic cell 
division of spermatocytes into spermatids. Enzymatic 
activity decreased remarkably after the differentiation of 
spermatids into spermatozoa. Enzymatic activity in the 
enriched Sertoli cells was 1.5 to 1.7 fold higher than in the 
enriched spermatogenic cells between day 15 and day 30 of 
age. The increase in synthase specific activity in 

spermatogenic cells and Sertoli cells indicates that both are 
contributing to changes in the enzymatic activity in 
seminiferous tubules. This change may be important in 

regulating the availability of DOL P for glycoprotein 
synthesis during early stages of differentiation. 

Spermatogenesis proceeds through a precise sequence of 
biochemical and morphological phases, spermatogonia!, meiotic 



95 

and spermatid (Leblond & Clermont, 1952). During the second 
and third phases, many immunological and biochemical changes 
occur which are unique to the spermatogenic process. Among 
these changes are the elaboration of spermatogenic cell or 
Sertoli cell specific glycoproteins (Fenderson et al . , 1984; 
Parvinen, 1982). For example, the spermatogenic cells 

produce an acrosomal protease precursor, proacrosin, and 
unique cell surface glycoproteins, which are required for 
fertilization. Proacrosin is first produced during the 
spermatid stage of differentiation but is retained throughout 
the remainder of spermatogenesis (Florke et al . , 1983). 
Other proteins are only expressed in specific stages, so that 
they may be absent or low in the spermatogonial and spermatid 
phases but are abundant during the meiotic phase. (e.g. 
fucosyl transferase in prepuberal mouse testes) (Letts et 
al., 1974a). 

The Sertoli cell, a non-germinal support cell of the 
seminiferous tubule, plays a critical role in the 
spermatogenic process by providing structural support, 
regulating spermatogenic cell movement, and compartmentation 
of spermatogenic cells from non-germinal cells, as well as 
mediating the movement of hormones, metabolites and nutrients 
to and from the developing spermatogenic cells (Ritzen et 
al . , 1981). The Sertoli cell produces many glycoproteins 
that support these functions including androgen binding 
protein (Parvinen, 1982) and plasminogen activator (Lacroix 



96 

et al . , 1977). Coordinated secretion of androgen binding 
protein by the Sertoli cell and the association of the 
Sertoli cell with pachytene spermatocytes during the meiotic 
phase even suggests the possibility of intercellular 
communication in regulating temporal expression of certain 
Sertoli cell proteins (LeMagueresse et al . , 1980; Ritzen et 
al., 1982; Galdieri, 1984; LeMagueresse & Jegou, 1986). 

The biosynthesis of these glycoproteins at specific 
phases during testicular development requires a coordinate 
functioning of a series of enzymes, so that cofactors and 
associated biochemical apparatus must be present in the cell 
at or before the time of glycoprotein expression or function. 
However, only a few studies have described the regulation of 
expression of these components as possible controlling 
factors of spermatogenesis. 

Three of the glycosyl transferases, which are needed 
for the terminal reactions in glycoprotein oligosaccharide 
biosynthesis in rat and mouse testis, have been shown to 
increase in specific activity in a sequential manner, that 
parallels their use in the last steps of oligosaccharide 
processing (Letts et al . , 1974a). 

The availability of DOL P, another critical component 
in the synthesis of the N-linked glycoproteins, has been 
suggested as a r ate - 1 imi t ing factor in some developmental 
processes (Lucas, 1979; Harford & Waechter, 1980; Rossignol 
et al., 1980). Therefore, the study of DOL metabolism will 



97 

be undoubtedly useful for the research of glycoprotein 
regulation. James and Kandutsch (1980c) reported that mouse 
spermatogenic cells were more active than liver cells in DOL 
biosynthesis. Furthermore, Potter et al . (1981b) identified 
the pachytene spermatocytes as one of the most active 
spermatogenic cells in DOL synthesis. This prompted our 
study of dehydro DOL PP synthase, an enzyme that could 
contribute to increased DOL levels during spermatogenesis. 

Chapter II described In vitro assays for the synthase, 
which were developed to measure changes in the potential of 
seminiferous tubules to biosynthes ize dehydro DOL PP, a 
probable precursor in the biosynthesis of DOL P and DOL. The 
temporal expression of synthase correlated well with the 
increase of DOL P measured by HPLC methods in seminiferous 
tubules during early stages of differentiation in prepuberal 
rats. It was proposed, therefore, that the level of DOL P in 
rat seminiferous tubules might be controlled by the 
regulation of d_e novo dehydro DOL PP biosynthesis. 

In the previous chapter, the specific activity of the 
dehydro DOL PP synthase was shown to fluctuate during 
testicular development; it was hypothesized that a difference 
in enzymatic activities during development might due to the 
presence of different cell populations with different 
enzymatic activities in rat testes. In this chapter, the 
methods of cell fractionation are described, the specific 
activities of the dehydro DOL PP synthase are measured in 



98 

different purified spermatogenic cell populations and in 
Sertoli cells. A model is presented which explains the time 
dependent change of the dehydro DOL PP synthase specific 
activity in tubules of prepuberal rats. 



Materials and Methods 



Materials . Sprague - Dawley rats were obtained from 

local suppliers. t,t-Farnesyl diphosphate was prepared as 
previously described (Baba & Allen, 1978). [ 14 C]-A 3 - 

Isopentenyl diphosphate was purchased from Amersham/Searle 
Corp. Bovine serum albumin (BSA), trypsin, trypsin 

inhibitor, deoxyr ibonuclease , and collagenase were purchased 
from Sigma Chemical Corp. All other chemicals were of 
reagent grade . 

Solutions . Phosphate buffered saline, essential amino 
acids (BME 50X) and MEM nonessential amino acids were 
obtained from Gibco Labs. Enriched Krebs-Ringer bicarbonate 
medium (EKRB) contained 120.1 mM NaCl, 4.8 mM KC1, 25.2 mM 
NaHC0 3 , 1.2 mM KH2PO4, 1.2 mM MgS0 4 -7H 2 0, 1.3 mM CaCl 2 , and 
was enriched by the addition of 11 mM glucose, 1 mM 
glutamine , 10 ml/liter of essential amino acids, and 10 
ml/liter nonessential amino acids. Streptomycin sulfate (100 
Hg/ml) and penicillin G (K + salt) (60 /xg/ml) were also added 
to the medium. The solution was prepared from a stock 
solution immediately prior to use, filtered (0.30 p 



99 

Millipore), and the pH adjusted to 7.3 by a 15-20 min 
aeration with 5% CO2 in air. Glassware and other equipment 
was siliconized before use in order to reduce damage and 
adhesion of cells. 

Preparation of cell suspensions . Rats aged 7-65 days 
were sacrificed and testes were removed as described in 
Chapter II. Testicular cell suspensions were prepared by a 
modification of the two-step enzymatic method described for 
mouse (Romrell, 1979). The decapsulated testes were placed 
in a 50-ml Erlenmeyer flask containing 20 ml of collagenase 
(1 mg/ml) and DNAse (1 /ig/ml) in EKRB . The testes were 
incubated at 33°C in a shaking water bath operated at 120 
cycles/min, until the seminiferous tubules were freely 
dispersed in the incubation medium (10-15 min). The 
dispersed seminiferous tubules were allowed to sediment and 
the supernatant was decanted. The isolated tubules were 
washed twice with EKRB. Then fresh EKRB (20 ml) containing 
trypsin (2.5 mg/ml) and DNAse (1 /ig/ml) was added to the 
tubules and this suspension was incubated for 15 min in a 
shaking water bath as just described. The resulting cell 
suspension was gently pipetted approximately 50 times with a 
Pasteur pipet. Trypsin inhibitor (2.5 mg/ml) was added, and 
then the cell suspension was mixed with 10 ml of 0.5% BSA in 
EKRB and centrifuged at 200 x g for 10 min. The resulting 
pellet was washed three times with EKRB containing 0.5% BSA 
and 1 /ig/ml DNAse and resuspended in the same solution after 



100 

the washing. The suspension was filtered through a nylon 
mesh (135 /i) to remove cell aggregates. The cell 

concentration was determined using a hemocy tometer . This 
enriched spermatogenic cell suspension was finally suspended 
in EKRB containing 0.5% BSA and adjusted to a concentration 
of 2 x 10 6 cells/ml. 

Spermatogenic Cell Fractionation . The entire cell 

separation procedure was carried out at 5° C. Spermatogenic 
cells were separated by a modification of the STA-PUT unit 
gravity procedure (Romrell et al . , 1977). The sedimentation 
chamber was initially filled with 70 ml EKRB. Then the cell 
suspension, which contained 10° cells in 50 ml of 0.5% BSA in 
EKRB, was introduced into the chamber at a flow rate of 10 
ml/min. The sample was followed by a linear gradient of 2% 
to 4% BSA in EKRB generated from two interconnected 
reservoirs, that contained 1100 ml of 4% BSA and 1100 ml of 
2% BSA, respectively (total volume 2200 ml). Five min after 
loading the cell suspension, the flow rate was increased to 
40 ml/min. Eighty minutes after loading the cell suspension, 
the chamber was drained in 10-ml fractions at a rate of 10 
ml/min. Cell collection was finished within 5 hr after 
introducing the cell suspension to the chamber. The 
separated cell fractions were numbered and centrifuged at 200 
g for 10 min; the supernatant were decanted; the resulting 
pellets were resuspended in 0.5 ml of EKRB. Aliquots were 
taken from individual samples and checked for cell type and 



101 

purity. The cells were examined by Nomarski differential 
interference and phase microscopy. The samples of enriched 
early spermatids (stages 1 through 8) and pachytene 
spermatocytes were pooled separately, washed three times with 
phosphate buffered saline solution and used immediately for 
the measurement of the enzymatic activity. The cellular 
purity of the pachytene spermatocyte (Fig. 3-1-A) was 70%; 
the major contaminants being Sertoli cells and spermatids. 
The purity of the spermatid (Fig. 3-1-B) fractions was 80%, 
with pachytene spermatocytes, the primary contamination. 

Spermatozoa were obtained from adult rats (3 months of 
age). The cauda epididymis was removed and flushed with 1 ml 
of phosphate buffered saline via the ductus deferens. The 
collected spermatozoa were then washed three times in 
phosphate buffered saline and used for the enzymatic assay. 

Sertoli Cell Preparation . Sertoli cells were prepared 
by modification of the procedure of Dorrington et al . (1975). 
Sertoli cells were isolated from Sprague - Dawley rats of 
specific ages. Tubules were treated with proteases as 
described above except that phosphate buffered saline was 
used in place of EKRB and more (3 /ig/ml) DNAse was used. The 
process was similar to that for s p e r ma t o genie cell 
fractionation, only the Sertoli cells retained on the nylon 
filter were collected as the fraction enriched in Sertoli 
cells. These aggregates of 10-50 Sertoli cell was further 
treated for 3-4 min with a hypotonic solution of two-fold 



102 



diluted phosphate buffered saline to remove spermatogenic 
cells. Under these conditions the spermatogenic cells are 
lysed but the Sertoli cells retain their integrity. The 
cells were examined by Nomarski differential interference and 
phase microscopy to check for purity and integrity. The 
resulting cell suspension contained more than 80% Sertoli 
cells with a contamination of pachytene spermatocytes and 
spermatids (Fig. 3-1-C). 

Homogenate Preparation . Homogenates were prepared from 
seminiferous tubules, cell suspensions from tubules, enriched 
spermatogenic cell populations and Sertoli cells. The 
excised testicular tubules were weighed, suspended at a ratio 
of 1:2 (w/v) in ice cold buffer (20 mM Tris-HCl pH 7.5 and 1 
mM EDTA) and sonicated as described in Chapter II. 
Homogenates of various mixed and enriched cell fractions, 
were prepared similarly, except that in these cases the 
packed cells were suspended in two volumes of buffer (v/v) . 
Protein quantitation was determined by the method of Lowry et 
al . (1951) before assay . 



•'-The cellular dissociation procedure used a relatively 
large amount of BSA (2-4%). There was a concern that binding 
of BSA to the purified cell fractions might lead to some 
error in protein determination of these isolated cells. 
Therefore, it was useful to estimate the binding of BSA to 
the enriched eerm cells. Measurement of binding was carried 
out with [*"I]-BSA as a probe in 2% BSA. Protease 
dissociated germ cells were incubated with [^-^^I]-BSA for 3 
min at room temperature then washed. The radioactivity on 
the cell in the beginning and the end of the test was 
compared. The results showed negligible non-specific binding 
of BSA to the cell surface (144 jig/14350 fig which is less 
than 1%) after the STA-PUT fractionation and the buffer 



103 

Dehydro POL PP Synthase Assay . The enzyme activity was 
determined by the same assay method described in chapter II. 
The enzyme protein (1 mg) from homogenates of enriched cell 
populations or various cell mixtures were assayed as 
indicated. The products, dehydro DOL PP and dehydro DOL P 
were extracted with CHC1 3/CH3OH (2:1), isolated by TLC and 
quantitated as described in Chapter II. The level of 
enzymatic activity was expressed as the sum of the pmoles of 
isopentenyl diphosphate i n c o r po r a t e d/mg protein. The 
relationship of these two products were extensively discussed 
in the previous chapter. 



Results 



Synthase Activity in Protease Treated Seminiferous 
Tubules . The synthesis of dehydro DOL PP and dehydro DOL P 
from farnesyl diphosphate and [ l^C ]- isopentenyl diphosphate 
was compared in sonicates of protease - treated and untreated 
tubules from rats aged 7-65 days (Fig. 3-2). It is necessary 
to determine if the enzyme in mixed tubular cell populations 
has the same enzymatic properties as that in the isolated 
tubules. In another words, did proteases treatment change 
the enzymatic properties of the separated tubular cells? 
The fluctuation in enzyme specific activities in the protease 

washes. Therefore, the enzymatic activities measured in the 
study do not need to be corrected. 



104 

treated tubules was parallel to that seen in the untreated 

tubules. Although there was a decrease in specific activity 

in the protease treated tubules at all ages, there was no 

o 

apparent selective loss of activity at any particular age . 

Synthase Activity in Enriched Cells from Testis . The 
level of synthase activity was also evaluated in isolated 
spermatogenic cells and epididymal spermatozoa. The time 
course of formation of dehydro DOL PP and dehydro DOL P was 
evaluated in the enriched pachytene spermatocytes, spermatids 
and Sertoli cell (Figure. 3-3). The results indicated that 
the sum of product formation with 1 mg of protein from 
homogenates of each cell type increased linearly with 
increasing incubation time as shown in the chapter II for the 
tubular homogenates. Protease treatment did not have an 
adverse effect on the linearity of time dependent assay. The 
enzyme specific activity in Sertoli cell was the highest. 
Homogenates of cell fractions highly enriched for pachytene 
spermatocytes (70% purity) and spermatids (80% purity) had 
synthase activity equal to or higher than a whole testicular 



The methodology for dissociating cells from 
seminiferous tubules was satisfactory, since 80-90% of the 
enzymatic activity still remained after the proteases 
treatment. Dispase, a commercial preparation of collagenase 
has also been reported to dissociate several tissues 
successfully. Therefore, dispase was tested as a replacement 
for collagenase in the cellular dissociation from 
seminiferous tubules. About 40% of the enzymatic activity in 
the collected dissociated cells was lost by this treatment 
(results are not shown). 



Figure 3-1. Purity of Enriched Cell Fractions. 

The testicular tubules were dissociated with 
proteases and the spermatogenic and Sertoli cells were 
separated as described in the Materials and Methods. 
The purity of each cell fraction, as estimated by 
Nomarski differential interference microscopy (x 600), 
was estimated to be 80% for spermatids (Panel A), 70% 
for pachytene spermatocytes (Panel B) and 70% for 
Sertoli cells (Panel C) . 



106 




Figure 3-1. Panel A. Spermatids 



107 





Figure 3-1. Panel C. Sertoli Cells (continued) 



Figure 3-2. Dehydro Dolichyl Diphosphate Synthase 

Activity in Sonicates of Tubules from Rats of Different 
Ages . 

The enzymatic activity was assayed under standard 
conditions with sonicates of seminiferous tubules 
treated ( o ) and untreated ( • ) with protease as 
described in the Materials and Methods. The da^a is 
presented as the mean + standard deviation (x + ^-jj- ) . 
Numbers in parentheses indicate the number of animals 
used to prepare the tubules. 



Figure 3-3. Time Course of Incorporation of [ C]- 
Isopentenyl Diphosphate into Dehydro DOL PP and Dehydro 
DOL p . 

Incubations containing 100 mM Tris-HCl buffer 
(pH7.5), 10 mM MgCl 2 , 0.5% Triton X- 100, 250 fiU t,t- 
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, 36 /jM f 1 - 
l^C ] - i s openteny 1 diphosphate, and 1.0 mg protein of 
sonicated of enriched pachytene spermatocyte, spermatid, 
and Sertoli cell from seminiferous tubules as enzyme 
protein in a total volume of 0.25 ml were carried out at 
37° C for the indicated times. The sum of [ ]- dehydro 
DOL PP and [ 14 C ]- dehydro DOL P were estimated by the 
method described before. Sertoli cells are isolated 
from 23 day old rats, spermatogenic cells are from 40 
day old rats . 



112 




113 

homogenate with or without protease treatment (Fig. 3-4). On 
the other hand, the spermatozoa and spermatogonial enriched 
cell fractions had activities less than 20% of the 
spermatocyte activity. When the enzymatic activity was 

expressed as pmoles/mg protein/hr, pachytene spermatocytes 
had an activity 1.6 fold higher than that seen with a mixture 
of cells obtained from protease treated seminiferous tubules. 
The enzymatic activity of the enriched spermatocytes was 
about 1.4 fold higher than enriched spermatids, 4.8 fold 
higher than spermatogonia and about 7.6 fold higher than 
spermatozoa. The enzymatic activity of the spermatogenic 
cells may also be expressed as pmoles product formed/10^ 
cells/hr (Table 3-1). In this case the enzymatic activity of 
spermatocytes (28.9 pmoles/10" cells/hr) was 4.5 fold higher 
than that of spermatids (6.5 pmoles/10^ cells/hr) and about 
126 fold higher than that of spermatozoa (0.23 pmoles/10 6 
cells/hr) . 

Synthase Activity in Sertoli Cells . Synthase specific 
activities were also measured in homogenates of Sertoli cells 
and a mixed spermatogenic cell population from rats 7-65 days 
old (Fig. 3-5). The fluctuations in the enzyme specific 
activities for both of these cell populations were parallel 
to that seen with the protease and non-protease treated 
tubules. In each case, the activities peaked at day 23. The 
enriched Sertoli cell specific activity ranged from 1.5-2.3 



114 

fold higher than that of the mixed spermatogenic cell 
population between day 14 and day 30. 

Estimated Activities in Pure Cell Populations . 
Synthase specific activities in "pure" populations of the 
different spermatogenic cells and the Sertoli cells were 
estimated from the known purity of the enriched cell 
fractions and their specific activities. Table 3-2 shows 
that, after a correction was made for the contamination of 
each cell fraction for other spermatogenic cells, the enzyme 
specific activity in pachytene spermatocytes was 1.7, 5.3 and 
8.3 fold higher than in spermatids, spermatogonia and 
spermatozoa, respectively. Estimates of the specific 

activities of "pure" Sertoli cells isolated from rats of 
different ages is shown in Table 3-3. There was a 4.5 fold 
increase in the enzyme activity of the Sertoli cell between 
day 7 and day 23 and 4.3 fold decrease in activity from day 
23 to day 65. 

In summary: 1) dehydro DOL PP synthase activity in 
spermatogenic cells and Sertoli cells peaks in rats at age 23 
days, 2) the specific activity of the enriched and pure 
spermatogenic cell populations tested decreased in the 
following order: pachytene spermatocyte > spermatids > 
spermatogonia > sperm and 3) synthase specific activity for 
Sertoli cells was 1.5-1.7 fold higher than that of 
spermatogenic cells in rats between day 15 and day 30 of age. 



Figure 3-4. Dehydro Dolichyl Diphosphate Synthase 

Activity in Enriched Spermatogenic Cell Population. 



Synthase activity was measured in sonicates of 
seminiferous tubules treated and untreated with 
protease, spermatogonia enriched spermatogenic cells 
from tubules of 7 day old rat, pachytene spermatocytes, 
spermatids (from 40 day old rats) and epididymal 
spermatozoa (from 3 months old rats). The specific 
activity of the enzyme is presented as the sum of pmoles 
of dehydro DOL PP and dehydro DOL P formed/mg protein 
hr . 



116 




117 



Table 3-1 



Dehvdro Dolichvl Diphosphate Synthase Activity 


in 




Enriched 


Spermatogenic Cells 3 




Experiment 




Activity 


Ratio 




pmoles/10 6 Cell ■ hr 






Pachytene (P) 


Spermatid (S) 


P/S 


1 


34.9 


7.9 


4.4 


2 


33.1 


7.9 


4.1 


3 


22.9 


5.9 


3.9 


4 


25.0 


4.5 


5.6 


Average 


28.9 ± 2.9 


6.5 ± 0.8 


4.5 ± 0.4 



a Enzyme activity was measured in cellular sonicates as 
described in the Materials and Methods and expressed as pmoles of 
isopentenyl diphosphate incorporated per 10^ cell per hour. The 
cell numbers were calculated by using the conversion factors 258 fig 
protein = 10 6 cells and 83 /jg protein - 10 6 cells, for pachytene 
spermatocytes and spermatids, respectively (from L. J. Romrell's 
unpublished data) . 



Figure 3-5. Age Dependent Variation in Synthase 

Activity in Sertoli Cells, Spermatogenic Cells and 
Protease Treated Seminiferous Tubules. 



Synthase activity was measured under standard 

conditions in sonicates of Sertoli cells ( o ) , 

spermatogenic cells filtrate from protease treated 

seminiferous tubules ( A ) , and protease treated 

seminiferous tubules ( • ) prepared from rats of 
different ages. 



119 



140 




i i i i i 1 1 1 1 ' 1 — — ■ ■ ■ 

10 20 30 40 50 60 70 

Days After Birth 



120 



Table 3-2 

Estimated Specific Activities of Dehydro POL PP Synthase In 
Pure Spermatopenic Cells a 



Specific Activity (pmoles/mg protein) 
Spermatogenic Cell % Cell Purity Enriched "Pure" 



Spermatogonia 100 19 19 

Pachytene 80 91 100 
Spermatocytes 

Spermatid 85 64 58 

Spermatozoa 100 12 12 



a Specific activities for pachytene spermatocytes and spermatids were 

estimated by assuming that each of these populations was contaminated 

by the other cell population. The two simultaneous equations 

SA = Fraction Pachytene (SA , ) + Fraction SpermatidfSA ) 

_ Enriched . , , . * \. pach , r ,\ sper 

tor the enriched populations of spermatocytes and spermatids were 

solved to obtain the specific activities of both pure cell 

populations . 



121 



Table 3-3 

Estimated Specific Activities of Dehydro POL PP Synthase in "Pure" 
Sertoli Cells From Rats of Different Ages a 



Specific Activity (pmoles/mg Protein) 

Day of %Purity of Enriched Enriched "Pure" 

Age Sertoli Sertoli Spermatogenic Sertoli 

Cells Cells Cells Cells 



7 


87 


27 


19 


28 


14 


88 


109 


47 


118 


23 


88 


119 


69 


126 


30 


86 


89 


61 


94 


40 


85 


63 


49 


66 


65 


81 


30 


25 


29 



a Specific activities of "pure" Sertoli cells were estimated by 
assuming that each of the enriched Sertoli cell populations was 
contaminated by the same mixture of spermatogenic cells as that 
isolated from rats of the same age. The following equation 
^Enriched Sertoli " Fracti ™ Se "oli cell (SA Ser ) + Fraction 

Germ cell I SA Enxi ^ ed ) was solved to obtain the specific 

activity for each ff pure" g< Sertoli cell population. 



122 



Discussion 



Glycoproteins are undoubtedly critical components in 
the spermatogenic process. The timing of their elaboration 
is thought to be critical for spermatogenesis to proceed 
normally. Histochemical studies in spermatogenic cells show 
that the Golgi apparatus of spermatocytes and early 
spermatids are highly active in glycoprotein biosynthesis 
(Letts et al., 1974b). However, the Golgi apparatus 

degenerates and is lost from spermatogenic cells as 
spermatids mature into spermatozoa. At least some of the 
enzymes necessary for N - g 1 y c o pr o t e in biosynthesis are 
presumably lost as well. Letts e_t a_l (1974a) have shown 
developmental dependent changes in the specific activities of 
three glycosyl transferases involved in the late reactions of 
oligosaccharide maturation in glycoprotein biosynthesis. 
Galactosyl and N- acetylglucosaminyl transferases were found 
in spermatogonia, whereas the fucosyl transferase was not 
highly active until the spermatocytes appeared. All three 
enzymes were low in spermatozoa. A later study (Letts et 
al . , 1978) indicated that spermatocytes and early spermatids 
were highly active in glycoprotein biosynthesis. Therefore, 
it was of interest to investigate if other biological 
machineries necessary for the glycoprotein biosynthesis are 
changing accordingly in these cell populations. 



123 

The timing of the secretion of glycoproteins such as 
androgen binding protein and plasminogen activator by the 
Sertoli cell, has been described in several laboratories 
(Parvinen, 1982). Furthermore, there are now numerous 

reports which show that the secretion of androgen binding 
protein may be regulated by the type of spermatogenic cells 
associated with the Sertoli cell at different times during 
the spermatogenic cycle (LeMagueresse et al . , 1980; Ritzen et 
al., 1982; Galdier, 1984). 

Recent studies indicated that the mammalian testis 
exhibits unusually high rates of DOL synthesis. This could 
be related to high rates of glycoprotein biosynthesis, and to 
temporally regulated synthesis of acrosomal enzymes in late 
pachytene spermatocytes or early spermatids (James & 
Kandutsch, 1980c; Wenstrom & Hamilton, 1980; Potter et al . , 
1981b). Acrosomal enzymes may also represent end products of 
dolichol -mediated glycosylat ion in the testis since many of 
these constituents are glycoproteins (Flechon, 1979; Mukerji 
& Meizel, 1979). Since DOL P has such a critical role in N- 
linked glycoprotein biosynthesis, its availability is also a 
potentially regulatory factor of glycoprotein biosynthesis 
during spermatogenesis. The results of previous work (Allen 
& Ward, 1987; Chapter II) showed that the level of DOL P and 
dehydro DOL PP synthase increased in parallel between day 7 
and day 23 of spermatogenesis in the seminiferous tubules of 
immature rats. Synthase activity then decreased to a level 



124 

at day 60 near that seen at day 7 . The increase in DOL P 
levels is consist with the observations of Nyquist and Holt 
(1986), who showed an increase in DOL concentrations in rat 
testes during this time period and the work of Potter et al . 
(1981b), who showed a high rate of DOL biosynthesis from 
acetate by mouse pachytene spermatocytes. Potter and 

coworkers also showed that hydr oxyme thy 1 glutaryl CoA 
reductase activity was high in pachytene spermatocytes. 
Nyquist and Holt (1986) have also reported that DOL 
concentration was high in Sertoli cells and suggested that 
DOL may be synthesized in the spermatogenic cell then 
transported to and accumulated in the Sertoli cell. This 
conclusion was supported by the observation by James and 
Kandutsch (1980c) that testes of x-irradiated mice or testes 
of mutant mice severely deficient in spermatogenic cells (but 
with apparently normal Sertoli cells) incorporated acetate 
into DOL at a 20 fold lower rate than normal testes. 

However, the results reported here show that both the 
spermatogenic cells and the Sertoli cells contribute 
substantially to the dehydro DOL PP synthase activity of rat 
seminiferous tubules during early stages of testicular 
development. This is not surprising considering the active 
glycoprotein biosynthesis occurring in both cell types, the 
rapid changes occurring in the spermatogenic cell during 
differentiation and the active role of the Sertoli cell in 
supporting this development. 



125 

Assignment of the relat ive contribution of the 
spermatogenic cells and the Sertoli cells to the changing 
synthase specific activities at different times during early 
spermatogenesis requires an assessment of the fraction of 
each of these cells present at different ages and knowledge 
of the specific activities of the "pure" cell types. An 
interpretation of these changes is made here on the basis of 
the reported results (Fig. 3-6). 

At 7 days of age the synthase activities of both the 
Sertoli and spermatogenic cells are low. At this stage of 
differentiation the Sertoli cells are still dividing and the 
spermatogenic cells have not yet reached the meiotic phase. 
It is reasonable that the observed synthase activity from 
testes at this age was low. 

At 15 days of age, the Sertoli cells have stopped 
dividing (Steinberger and Steinberger, 1971) and at least 
some of the spermatogenic cells have entered the meiotic 
phase. Dehydro DOL PP synthase in spermatogenic cells has 
increased but the cell number is small. Therefore, 
spermatogenic cells do not contribute in a major way to the 
total specific activity of the synthase in the whole tubules. 
In contrast the Sertoli cells are relatively high in both 
number and in enzyme specific activity. Therefore, the total 
tubular synthase activity is mainly due to Sertoli cells. 

At 23 days of age, the enzyme specific activities of 
both early pachytene spermatocytes and Sertoli cells have 



126 

peaked. The number of pachytene cells relative to other 
spermatogenic cells is maximum, and there are few if any 
spermatids present by this time. The number of Sertoli cells 
has become relatively constant. Therefore, the total 

activity of synthase is due to the sum of Sertoli cell, 
pachytene spermatocytes and other spermatocytes preceding the 
pachytene stage. The specific activity is optimal at this 
time, because Sertoli cell specific activity is highest at 
this point and the relative percentage of pachytene 
spermatocytes (the spermatogenic cell with the highest 
specific activity) is also highest at this time of 
development. Actually, it is the only time period in the 
rat's life span that the relative percentage of pachytene 
spermatocytes reaches a peak value (Fig. 3-6). 

At 30 days of age, the pachytene spermatocytes still 
have active synthase activity but the relative number of 
these cells present in the seminiferous tubules is a smaller 
fraction of the total spermatogenic cell population than at 
day 23. The Sertoli cell number is constant but its synthase 
specific activity has decreased by day 30. The contribution 
of spermatids to the total activity is relatively low at this 
time, since the number of spermatids is small although 
increasing rapidly. Our data also indicate that as pachytene 
spermatocytes differentiate into spermatids, the enzyme 
specific activity decreases by 30 % (Fig. 3-4). There was, 
however, no net change in total enzyme activity during the 



127 

division of pachytene spermatocytes into spermatids (Table 3- 
1). Most of the enzymatic activity is lost during the 
differentiation of spermatids to spermatozoa. Therefore, the 
decrease in tubular synthase activity after 23 days can be 
accounted for by the decreasing fraction and specific 
activity of Sertoli cells and the fact that the pachytene 
spermatocytes, which have the highest spermatogenic cell 
specific activity, are becoming a smaller fraction of the 
total spermatogenic cells population and are being replaced 
by spermatids with lower specific activity. 

Kumari and Duraiswami (1986) have estimated the 
percentages of Sertoli and spermatogenic cell populations in 
rat seminiferous epithelium at various days during early 
stages of spermatogenesis. If these percentages are used in 
conjunction with the enzyme specific activities observed here 
for the Sertoli and mixed spermatogenic cell population, the 
predicted enzymatic activities of the whole tubular 
homogenate can be calculated for animals 15, 23, and 30 days 
of age (Fig. 3-6). There was a good correlation between the 
predicted specific activity and the observed activity from 
this study except for the result on day 15. 

In general, the specific activity of dehydro DOL PP 
synthase in Sertoli cell was always higher than that in 
spermatogenic cells measured between day 7 and day 65 of age. 
This suggests that glycoprotein biosynthesis is more active 
in Sertoli cell than in spermatogenic cells. Kumari and 



Figure 3-6. The Relationship between the Dehydro DOL PP 
Synthase Activity and Spermatogenesis during Testicular 
Deve lopment . 

The curve of the enzymatic activity of rat 
testicular development is from the results in the 
previous chapter. The scheme is modified from B. P. 
Setchell., 1982, in Germ Cell and Fertilization, Eds. C. 
R. Austin and R. V. Short, New York: Cambridge 
University Press, PP. 63-101. The calculated results 
are shown as ( A ) . 



129 




DAYS AFTER BIRTH 



130 

Duraiswaml (1986) have concluded, based on an 
autoradiographic study, that the protein synthetic potential 
of Sertoli cells is greater than that of spermatogenic cells 
at any stage of differentiation. The parallel results seen 
between that study and results represented here may be highly 
significant and suggest a key role for dehydro DOL PP 
synthase in regulating glycoprotein biosynthesis in Sertoli 
cells during spermatogenesis in rats. 

The reason for the rise and fall of the enzyme specific 
activity in Sertoli cells in the immature testes is not well 
understood. Enzyme activities and protein secretion in 
rodent seminiferous tubules have been shown to peak at about 
day 20 to day 23 in several cases ( Parvinen , 1 9 8 2 ) . This has 
been attributed in some cases to the appearance of pachytene 
spermatocytes with their constituent enzymes and in other 
cases to the onset of new protein synthesizing activities in 
Sertoli cells. In the current study, the results showed that 
both Sertoli cell and spermatogenic cells are contributing to 
the increase of the dehydro DOL PP synthase activity. The 
possibility that the presence of the pachytene spermatocyte 
may cause an increase in Sertoli cell synthase activity is an 
interesting conjecture, which has been postulated for other 
systems (LeMagueresse & Jegou, 1986; Ireland & Welsh, 1987) 
and requires further study. 



CHAPTER IV 
CONCLUSIONS AND DIRECTIONS 



Dehydro DOL PP synthase, which catalyzes the synthesis 
of dehydro DOL PP from farnesyl diphosphate and isopentenyl 
diphosphate could be very important for controlling DOL P 
level in eukaryotic cells, since this is the only de novo 
biosynthesis pathway known and serve as a "bridge" connecting 
the small metabolites, such as acetyl CoA, with the large DOL 
molecules. Therefore, dehydro DOL PP synthase could be an 
important cellular regulator of glycoprotein biosynthesis as 
a consequence of its regulation in the DOL P de novo 
biosynthesis . 

An attempt has been made to further characterize the 
enzymatic products, namely, dehydro DOL PP and dehydro DOL P. 
The precursor-product relationship between dehydro DOL PP and 
dehydro DOL P seems clearly established. 

It has been reported that the level of DOL P, a 
carbohydrate carrier in glycoprotein biosynthesis, is 

regulated during spermatogenesis (James & Kandutsch, 1980b; 
Potter et al . , 1981b). Temporal expression of seminiferous 
tubular dehydro DOL PP synthase has been shown to correlate 
well with the increase in Dol P during early stages of 
differentiation in prepuberal rats. The results presented in 

131 



132 

Chapter II support the hypothesis that increasing DOL 
synthesis observed during testicular development (Potter et 
al . , 1981b) is due at least in part to an increase in the 
dehydro DOL PP synthase activity. 

The cellular localization of this increased synthase 
activity was of interest because of the multicellular nature 
of the testicular tubules. The specific activity of synthase 
in homogenates of protease treated seminiferous tubules, cell 
fractions enriched in spermatogenic cells or Sertoli cells 
peaked in rats aged 23 days, as shown with non-protease 
treated cells. Homogenates of cell fractions enriched in 
pachytene spermatocytes, spermatids or Sertoli cells had 
higher synthase activity than a whole testicular homogenate 
or a mixture of cells prepared by protease treatment of 
tubules. Enzymatic activity in pachytene spermatocytes 

expressed per mg protein, was about 5.3 fold higher than 
spermatogonia, 1.7 fold higher than in spermatids and about 
8.3 fold higher than in spermatozoa. Therefore, the increase 
of the synthase activity in spermatogenic cell before day 23 
can be accounted for by the appearance of the pachytene 
spermatocytes. Little net increase in enzyme occurred during 
or after meiotic cell division of pachytene spermatocytes 
into spermatids. The enzymatic activity decreased remarkably 
during the differentiation of spermatids into spermatozoa. 

It is reported for the first time that Sertoli cells 
have the potential to synthesize DOL P. The enzymatic 



133 

activity in enriched Sertoli cells was 1.5 to 2.3 fold higher 
than in the enriched spermatogenic cells between day 14 and 
day 30. The increase in synthase activity in spermatogenic 
cells and Sertoli cells indicates that both are contributing 
to changes in the enzymatic activity in seminiferous tubules. 
In addition, and perhaps more significantly, the work 
presented here provides evidence that dehydro DOL PP synthase 
may be important in regulating the availability of Dol P for 
glycoprotein synthesis during early stages of spermatogenesis 
in rat. 

Future research on the role that dehydro DOL PP 
synthase plays in spermatogenesis in rat will focus on 
several fundamental questions. In vitro studies on the 
incorporation of radiolabeled probe, such as mevalonate, 
into DOL with enriched Sertoli cells will determine whether 
synthase activity function in these cells in vivo . Synthase 
activity would also be measured in co-cultures of Sertoli 
cells and different spermatogenic cells. This could test 
the role of cell-cell interaction with respect to the 
function of DOL P in regulating spermatogenesis. Activators 
or inhibitors of this enzyme may also be produced as a 
result of cell-cell interactions. Such questions can now be 
approached with the techniques and information presented in 
this dissertation. 



APPENDIX A 



SUMMARY OF EXPERIMENTAL DATA PRESENTED IN FIGURE 2-13 DEHYDRO DOL PP 
SYNTHASE ACTIVITY IN SONICATES OF TUBULES FROM RATS OF DIFFERENT AGES. 



DATE 



AGE 



85/1/16 
2/5 
4/8 



4/22 
4/30 
5/16 
5/24 
6/10 



6/22 



7/3 



10 



14 15 23 28 30 



35 



50 60 64 



64.5 



72.9 



82.5 



77.3 
70.7 



51.3 



39.0 



97.2 



66.3 
62.9 
59.5 



85.2 

50.9 51.2 
50.4 44.4 
94.7 71.0 
34.3 
56.3 
67.0 



107.4 
90.2 
74.9 



106.0 
92.7 
92.7 



71.0 
73.2 
63.5 



58.7 
71.5 
60.7 
53.2 



71.7 
91.1 



100.7 
119.9 
110.0 
94.1 



73.3 
98.1 



59.9 
48.8 
74.8 



X 


70 


54 


61 


69 


89 


106 


87 


87 


69 


63 


59 


39 


Sx 


23 


14 


8 


6 


14 


11 


13 


14 


5 


3 


12 




N 


8 


12 


8 


4 


8 


8 


6 


14 


6 


6 


8 


2 


n 


4 


6 


4 


2 


4 


4 


3 


7 


3 


3 


4 


1 




12 


6 


4 


4 


7 


6 


7 


5 


3 


2 


6 




Animals 


40 


18 


8 


3 


10 


4 


5 


13 


3 


3 


10 


1 



Mean - x pmoles/mg protein 

Standard Deviation - S x //n" pmoles/mg protein 
Number of Experiments - n 
Number of Assays - N 

134 



APPENDIX B 



TYPICAL NUMBER OF RATS, TOTAL TUBULE WEIGHT, AND NUMBER OF ASSAY USED 
FOR EACH EXPERIMENT AS A FUNCTION OF RAT AGE 



Age 
(Days) 


# of Rats Used 


Total Tissue Weight 
(g) 


Assays 


3 


10 


0.09 


2 


7 


4 - 5 


0.12 


2 


15 


2 


0.20 


2 - 3 


23 


2 


0.85 


3 or more 


30 


2 


1.20 


3 or more 


40 


2 


1.60 


3 or more 


60 


2 


3.50 


3 or more 



135 



APPENDIX C 



SUMMARY OF EXPERIMENTAL DATA PRESENTED IN FIGURE 3-5 . AGE DEPENDENT VARIATION IN 
SYNTHASE ACTIVITY IN SERTOLI CELLS, SPERMATOGENIC CELLS AND PROTEASE TREATED 
SEMINIFEROUS TUBULES. SYNTHASE ACTIVITY IS PRESENTED IN UNITS OF PMOLES 
ISOPENTENYL DIPHOSPHATE INCORPORATED/MG PROTEIN 



Sertoli Tubules Spermatogenic 

Rats Assays Synthase Rats Assays Synthase Rats Assays Cells 
Age Activity Activity Synthetase 
(Days) Activity 



7 


24 


4 


27 


+ 


6 


12 


4 


32 


+ 


2 


24 


4 


19 


+ 


2 


15 


12 


4 


109 


+ 


4 


12 


6 


61 


+ 


14 


12 


4 


47 


+ 


7 


23 


7 


4 


119 


+ 


4 


7 


6 


93 


+ 


4 


7 


4 


69 


+ 


2 


30 


4 


4 


89 


+ 


8 


4 


6 


76 


+ 


7 


4 


4 


61 


+ 


2 


40 


6 


4 


63 


+ 


7 


6 


4 


52 


+ 


6 


6 


4 


49 


+ 


5 


60 


4 


4 


30 


+ 


3 


4 


4 


24 


+ 


1 


4 


4 


25 


+ 


3 



136 



137 



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BIOGRAPHICAL SKETCH 



Zhong Chen was born in Beijing, China, in 1944. In 
1962, Zhong began his preraedical education at Peking 
University. Later Zhong studied medicine at China Medical 
College (the former Peking Union Medical College founded by 
the Rockefeller Foundation) and he received the M.D. degree 
in 1968. After graduation he practiced medicine for several 
years and specialized in ophthalmology. In 1981, Zhong 
visited the Royal Hospital ( Ri gsho sp i tal e t ) and the 
University of Copenhagen in Denmark as a visiting scholar in 
the Departments of Ophthalmology and Virology. During that 
period of time, Zhong realized that biochemistry and 
molecular biology are the keys to open the mysterious kingdom 
of medicine. So he decided to brush up on biochemistry and 
use it as a tool to explore the unanswered questions in 
medic ine . 

After finishing his Ph.D., Zhong will move to Oklahoma 
City, Oklahoma, and the laboratory of Dr. Jordan J. N. Tang 
in the Oklahoma Medical Research Foundation to continue his 
training . 



146 



I certify that I have read this study and that in ray 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




Charles M. Allen, Chairman 
Professor of Biochemistry and 
Molecular Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




Biochemistry and Molecular 
Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




MftH 

ProfeVior of Biochemistry 
Molecular Biology 



and 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 



Thomas W. O'Brien 

Professor of Biochemistry and 

Molecular Biology 



I certify that I have read this study and that in ray 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




LyrinjJ . Rd^rell 
Professor of Anatomy and Cell 
B i o 1 o gy 



This dissertation was submitted to the Graduate Faculty of 
the College of Medicine and the Graduate School and was 
accepted as partial fulfillment of the requirements for the 
degree of Doctor of Philosophy. 

April, 1988 ^ 

Dean, College of Medicine 



Dean, Graduate School